Biology 6th HBOnline

TABLE OF CONTENTS
TABLE OF CONTENTS.............................................................................................. I 6TH HBONLINE......................................................................................................... 2 1. OBJECTIVES......................................................................................................... 2 2. INTRODUCTION TO MEIOSIS............................................................................... 3 3. MEIOSIS................................................................................................................. 4 4 COMPARISON OF MITOSIS AND MEIOSIS.......................................................... 8 5. SELF TEST............................................................................................................ 9 6. REPRODUCTIVE SYSTEM.................................................................................... 9 6.1. Structure of the Reproductive System...........................................................10 6.2. Male Reproductive System.............................................................................11 6.2.1. Male Structure..............................................................................................................11 6.2.2.Spermatogenisis and Hormonal Control........................................................................14 6.2.3. Histology of the Testis..................................................................................................16 6.3. Female Reproductive System.........................................................................16 6.3.1. Female Structure...........................................................................................................16 6.3.2. Oogenesis and Hormonal Control.................................................................................18 6.3.3. Histology of the Ovary..................................................................................................19 6.4.Birth Control and Infertility..............................................................................20 6.5. Self Test............................................................................................................ 22 7.DEVELOPMENT................................................................................................... 28 7.1.Objectives......................................................................................................... 28 7.2.Overview of Reproduction and Development.................................................29 7.3.Major Stages of Development..........................................................................29 7.4.Pregnancy Testing............................................................................................33 7.5.Birth Defects.....................................................................................................33 7.6.Congenital Malformation..................................................................................33 7.7.Development Card Game.................................................................................34 7.8. Development Websites....................................................................................40 8.INHERITANCE...................................................................................................... 40 8.1.Objectives......................................................................................................... 40 INHERITANCE, DNA AND FRONTIERS OF HUMAN GENETICS.......................... 40 8.2.Pedigrees.......................................................................................................... 41 8.3.A Virtual Molecular Genetics Lab....................................................................44 8.4.Self Test............................................................................................................. 46

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6TH HBONLINE 1. OBJECTIVES

Sexual reproduction starts with the production of two types of gametes (sex cells) through the process of meiosis. The production of sperm in the male through meiosis is called spermatogenesis. In females, the production of eggs is called oogenesis. The sperm swim to the stationary egg, a much large cell that contributes cytoplasm and organelles to the zygote. There are a large number of sperm deposited to ensure that a few find the egg so that one can fertilize it.

OBJECTIVES

After completing the lectures, HBOnline Unit 6 and the Unit 6 practical on the reproductive system you should be able to: describe and understand the process of meiosis define gamete, fertilisation and zygote explain the functional differences between male and female human gametes describe the overall anatomical structure of the male reproductive system give functions of the prostate gland, seminal vesicles and bulbo-urethral glands in the male reproductive system describe spermatogenesis understand the way in which sperm is nurtured during its brief locomotory period describe the functions in the male of the hormones testosterone, folliclestimulating hormone and luteinising hormone describe the hormonal control of sperm production and the maintenance of maleness during the life cycle describe the overall anatomical structure of the female reproductive system describe oogenesis construct an overview of the menstrual cycle list the major events of the human female 28 day menstrual cycle explain the hormonal control of this cycle understand the steps leading to, and the process of fertilisation understand what is meant by birth control and its methods document the major causes of infertility and how this might be overcome. ACTIVITIES To gain the most value from these exercises you must EXAMINE THE MODELS provided in the practical class. Introduction to Meiosis o Meiosis o Compare Mitotic and Meiotic Cell Divisions o Revision/Self-test Structure of the Reproductive System Male Reproductive System o Male Structure o Spermatogenisis and Hormonal Control Histology of the Testis*

Female Reproductive System o Female Structure o Oogenisis and Hormonal Control Histology of the Ovary* Birth Control and Infertility Self-test

The Reproductive System, Development, Inheritance 1.Objectives 2. Introduction to Meiosis 3.Meiosis 4.Comparison of Mitosis and Meiosis 5.Self Test

2. INTRODUCTION TO MEIOSIS
Read Seeley's Essentials of Anatomy and Physiology (7th ed.) Chapter 3 - Cell Structures and Their Functions Meiosis Chapter 19 - The Reproductive System Formation of Sex Cells Spermatogenesis Follicle and Oocyte Development The alternation between growth and division in a cell is called the cell cycle. There are two major phases in the cell cycle: the mitotic phase and interphase (a growth period). During the mitotic phase, two daughter cells that are identical to the parent cell are produced. If the parent cell is diploid, then the daughter cells are diploid (have two sets of chromosomes: 2n). Meiosis occurs as part of the cell cycle in tissues that produce gametes. When an organism produces gametes (sex cells) for sexual reproduction, they need to be haploid (have a single set of chromosomes: n), so that when the male and female gametes fuse during fertilisation the resulting zygote has the correct number of chromosomes (diploid: one haploid set from the father and one haploid set from the mother). Meiosis is the process by which the number of chromosome sets is reduced from diploid to haploid to form gametes. To achieve this, two consecutive cell divisions occur: Meiosis I and Meiosis II. These divisions result in four daughter cells, rather than the two daughter cells of mitosis, and each has only half as many chromosomes as the parent cell (all the daughter cells are haploid).

Term Mitosis Meiosis

Definition The division of the cell nucleus. Two daughter nuclei are produced that are identical to the parent cell. A two-stage division of the cell nucleus that results in four daughter cells with half the chromosome number of the parent

cell (daughter cells are haploid). These daughter cells are gametes (sex cells). Cytokinesi The division of the cytoplasm to form new separate daughter cells s immediately after mitosis/meiosis. Chromoso A gene-carrying structure found in the nucleus. Each chromosome me consists of a long DNA molecule. Chromatid One of two identical strands that make up a chromosome after replication during the S-phase of interphase. Homologo A pair of chromosomes that possess genes for the same us Pair characters at corresponding positions on each chromosome. One homologous chromosome is inherited from the father and one is inherited from the mother. Homologu One chromosome of an homologous pair. (In the early stages of e mitosis and meiosis each homologue appears as two chromatids) Diploid Haploid A cell containing two sets of chromosomes (2n): one from each parent. A cell containing one set of chromosomes (n).

Centromer The centralised region that joins two chromatids. Spindle fibres e also attach to this region during mitosis/meiosis. Centriole A structure in animal cells that produces the spindle during mitosis/meiosis. Two pairs of centrioles are present in cell division: one pair at each end of the cell.

View the animation Stages of Meiosis

3. MEIOSIS
Each chromosome consists of one DNA molecule(s) in early interphase (G1). During the S phase of interphase, the genetic material replicates so that each chromosome consists of two chromatids held together by the centromere.

View the animations of Meiosis, Crossing Over and Random Assortment of Chromosomes
Prophase I

Initially the chromosomes are not visible but they become progressively more visible as they shorten and thicken. Homologous chromosomes pair with each other. Each chromosome in a homologous pair is called ahomologue. In animal cells, the centrioles migrate to the poles during early meiosis and produce the spindle. The nuclear membrane gradually breaks down and disappears.

At early Meiosis I each homologous pair of chromosomes consists of 4 chromatids.

Metaphase I

The homologous pairs line up on the equatorial plane of the cell. Each homologue consists of two sister chromatids. The centromeres of the paired chromosomes are separate but the homologous pairs are still associated so the homologues line up in pairs. (In mitosis the homologous chromosomes do not pair and so line up independently of each other). The centromere of each homologue (joining the two chromatids) becomes attached to only one spindle fibre. Thus in each homologous pair the centromeres are attached to spindle fibres from opposite poles.

Anaphase I

The homologous chromosomes (each consisting of two chromatids) separate completely from each other and move towards their respective poles as the spindle fibres contract. Each pole receives only half the original number of chromosomes. Thus the first meiotic division results in two haploid daughter cells.

Telophase I and Cytokinesis

The homologous chromosomes (still consisting of sister chromatids) arrive at opposite poles in the cell and the nucleus separates to form two haploid daughter cells. The cytoplasm divides via cytokinesis, forming two haploid daughter cells. A brief interphase usually follows Meiosis I but there is no replication of DNA during this interphase. At the end of Meiosis I, each of the daughter nuclei are haploid. The Second Meiotic Division is similar to a mitotic one. There is no pairing of homologues in Meiosis II as they are located in separate cells. Prophase II A spindle forms in the new haploid daughter cells. Metaphase II Chromosomes line up along the equator of the spindle. This time each centromere attaches to spindle fibres from both poles. Anaphase II The centromeres divide so that single chromatids move to opposite poles. These chromatids are now called chromosomes and constitute a complete set of chromosomes for the new nucleus. Telophase II and Cytokinesis The spindle disappears, nuclear membranes form and cytokinesis occurs. The chromosomes uncoil, elongate and fade from view. The DNA becomes stretched out in the nucleus to allow gene expression during interphase.

The outcome of these two divisions (which constitute one complete meiotic division) is four haploid nuclei formed from one diploid nucleus. The four haploid cells produced by meiosis are the gametes i.e. sperm or eggs. At the end of telophase I each chromosome consisted of two DNA molecules. At the end of telophase II, each chromosome is now (There is no pairing of homologues in Meiosis II as they are located in separate cells.) DNA molecule(s). 1. In what ways do the nuclei formed at the end of meiosis II differ from the nucleus at the beginning of Meiosis I? (Hint: Meiosis I is often called reduction division.) Model Answer Nuclei are haploid not diploid, and each chromosome is now a single DNA molecule. Only one allele of each gene is present in each nucleus.

4 COMPARISON OF MITOSIS AND MEIOSIS

Read
Seeley's Essentials of Anatomy and Physiology (7th ed.) Chapter 3 - Cell Structures and Their Functions Table 3.3 Comparison of Mitosis and Meiosis

View the animation of Comparison of Meiosis and Mitosis

FEATURE

MITOSI S

MEIOSIS Divisio Division nI II

Halving the chromosome number Centromeres divide

absent present absent presen absent present t

Homologous chromosomes move to opposite poles absent present absent of spindle Pairing of homologous chromosomes Single chromatids (now chromosomes) move to opposite poles of spindle absent present absent presen absent present t

5. SELF TEST
Q1. How many daughter nuclei result from: a) one mitotic division? 2 b) one complete meiotic division? 4 Are the daughter nuclei genetically identical to each other after: a) mitosis? Yes b) meiosis? No Do the daughter nuclei differ genetically from the parent nucleus in: a) mitosis? No b) meiosis? Yes Q2.List six differences between meiosis and mitosis. MITOSIS MEIOSIS 1. 2 cells produced 4 cells produced 2. Diploid Haploid 3. Same as parent Different to parent cell 4. One division only Two divisions 5. No pairing Homologues pair 6. Daughter cells identical Daughter cells differ from each other

6. REPRODUCTIVE SYSTEM
Seeley's Essentials of Anatomy and Physiology (7th ed.) Chapter 19- Reproductive System Functions of the Reproductive System Formation of Sex Cells Male Reproductive System Female Reproductive System

6.1. Structure of the Reproductive System

1. 46 2. 2n 3. 46 4. 2n

10. Fertilisation 11. 46 12. 2n 13. Mitosis

5. 23 14. 46 Chromosomes 6. n 7. Meiosis 8. 23 9. n 15. 2n 16. Mitosis 17. 46 18. 2n

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6.2. Male Reproductive System

6.2.1. Male Structure

The Male Reproductive System 1

The Male Reproductive System 2

The Male Reproductive System 1

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The Male Reproductive System 2

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The Testis and Associated Structures

Structure Scrotal sacs Testis Testicular artery

Function contains and suspends the testes outside the body produces spermatozoa and the sex hormones supplies blood to the testis

Epididymis maturation and storage of sperm Seminal vesicles Prostate gland Penis Urethra adds mucous alkaline fluid to seminal fluid especially fructose (energy for sperm) prostatic fluids add substances to the seminal fluid which neutralise secretions in the vas deferens and vagina organ for the transfer of sperm to the vagina duct for the transfer of sperm and urine (temporal separation)

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6.2.2.Spermatogenisis and Hormonal Control

View the animation of Spermatogenesis

The process by which male gametes are produced is called spermatogenesis Male gametes develop stepwise through a number of stages. Complete the flow chart to show, in order, the names of the various stages of male gamete development. Spermatogonium primary spermatocyte secondary spermatocyte spermatid sperm Gonadotropin releasing hormone (GnRH) stimulates the secretion of FSH / follicle stimulating hormone and LH/ luteinising hormone Follicle stimulating hormone (FSH) is partially responsible for inducing spermatogenesis it stimulates the epithelium of the seminiferous tubules. Luteinising hormone (LH) stimulates the secretion of testosterone by the testes. Testosterone plays a part in development of male reproductive organs, secondary sexual characteristics, and spermatogenesis Spermatogenesis is controlled via a negative feedback mechanism involving the synthesis of the hormone inhibin. Seminal fluid is produced by the prostate gland, seminal vesicles and bulbourethral gland. 1. Describe the anatomy of human sperm. Model Answer The head contains the nucleus and acrosome, the midpiece has mitochondria in a spiral arrangement and the flagellum is the tailpiece. 2. List the events that occur with the maturation and storage of sperm before its release from the body. Model Answer 1 Sperm cells are produced in the seminiferous tubules. They move into the epididymis for maturation. At ejaculation, the sperm move into the vas deferens and fluids from the seminal vesicles and prostate are added to produce the semen. This moves down the ejaculatory duct into the urethra and is ejaculated. Model Answer 2 After their production, sperm cells are transported through the seminiferous tubules into a network of tubules in the testis, and then into the efferent ductules which carry sperm to the epididymis, where the sperm cells continue to mature (they develop the capacity to swim and bind to the secondary oocyte). Semen then travels along the vas deferens by peristaltic contractions. The vas deferens joins up with a short duct from the seminal vesicles at the prostate gland to form the ejaculatory duct, which extends into the prostate gland where it joins the urethra. The urethra extends from the urinary bladder

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to the distal end of the penis. Capacitation occurs after the ejaculation of semen into the vagina, prior to fertilization. 3. Describe the organs involved in the production of seminal fluid, list the fluids and their compositions and relate the functions of seminal fluid to the well-being of the sperm. Model Answer 1 The prostate gland produces an alkaline secretion which neutralises the acidic urine and vagina. Proteolytic enzymes make the semen more liquid. Seminal vesicles produce a mucus secretion containing nutrients for the sperm. The bulbourethral glands produce mucus to lubricate the urethra and vagina. Model Answer 2 The prostate gland produces a thin, milky, alkaline secretion (approx 30% of seminal fluid) that helps neutralise the acidic urethra as well as the acidic secretions of the testes, the seminal vesicles and vagina. The pH is important for sperm movement. These secretions also contain proteolytic enzymes that break down the coagulated proteins of the seminal vesicles and make semen more liquid. The seminal vesicles produce a thick, mucus-like secretion (approx 60% of seminal fluid) containing fructose and other nutrients to nourish the sperm cells. It also contains proteins that are thought to help destroy abnormal sperm cells. Prostaglandins, which stimulate smooth muscle contractions, are in high concentration and can cause contractions of the female reproductive tract. The bulbourethral glands and mucous glands of the urethra produce a mucous secretion (approx 5% of seminal fluid), which lubricates the urethra, providing a small amount of lubrication during intercourse and helps reduce the acidity in the vagina. 4. Describe the production and control of production of the regulating male hormone levels, including the role of FSH and LH in production of sperm and testosterone and the role of testosterone during the male life cycle. Model Answer 1 GnRH stimulates the production of FSH and LH. LH stimulates the production of testosterone by the testes. FSH stimulates sperm production. Inhibin inhibits FSH production. Testosterone affects sperm production, development of reproductive organs and secondary sexual characteristics. It also inhibits GnRH, LH and FSH production. Model Answer 2 Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus and targets the anterior pituitary gland, stimulating the secretion of luteinising hormone (LH) and follicle stimulating hormone (FSH). LH targets the interstitial cells of the testes and stimulates synthesis and secretion of testosterone. FSH targets the seminiferous tubules (Sertoli cells) of the testes and supports spermatogenesis and inhibin secretion. Inhibin is produced by Sertoli cells and targets the anterior pituitary, inhibiting FSH secretion through negative feedback. Testosterone is produced by the interstitial cells of the testes and targets the testes and other body tissues. It has multiple roles: development and maintenance of reproductive organs; support of spermatogenesis; development and maintenance of secondary sexual characteristics. Testosterone targets the anterior pituitary and hypothalamus, inhibiting GnRH, LH, and FSH secretion through negative feedback.

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6.2.3. Histology of the Testis

Label the diagrammatic representation of the testes.

6.3. Female Reproductive System

6.3.1. Female Structure

The Female Reproductive System 1

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The Female Reproductive System 2

Structure

Function

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Ovary

produces ova (egg cells) and sex hormones, oestrogen and progesterone

Oviduct/ receives ova from ovary; transports ova from ovary to (Fallopian tube) uterus; if sperm present fertilisation occurs in the upper third of oviduct Uterus Vagina site of implantation of a fertilised ovum; site of development of embryo to birth birth canal for expelling baby; copulatory organ which receives penis in copulation

The term urogenital (or urinogenital) system is more applicable to males than to females because in males the urethra is part of both systems, so the systems overlap in space, but not in time. In females the two systems, urinary and genital, are separate.

6.3.2. Oogenesis and Hormonal Control

The process by which female gametes are produced is called oogenesis. Female gametes also develop stepwise through a number of stages. Complete the flow chart to show, in order, the names of the various stages of female gamete development. Oogonium primary oocyte secondary oocyte ovum and polar bodies. Gonadotropin releasing hormone (GnRH) stimulates the secretion of FSH / follicle stimulating hormone and LH / luteinising hormone. Follicle stimulating hormone (FSH) stimulates follicular maturation and the secretion of oestrogen. Luteinising hormone (LH) stimulates final maturation of follicle, release of the ovum, and then the conversion of the ruptured follicle into the corpus luteum, with the subsequent production of progesterone. Oestrogen plays a part in the development of female reproductive organs and secondary sexual characteristics. Oestrogen also acts to maintain the hormone cycle and to stimulate proliferation in the endometrium. Progesterone stimulates maturation of uterine glands and maintains the endometrium in pregnancy-ready state. Ovulation is controlled through a negative feedback mechanism involving LH, FSH and the levels of oestrogen and progesterone acting on the follicles. The signal for menstruation to occur is a decline in progesterone and oestrogen levels caused by the degeneration of the corpus luteum. Progesterone and some oestrogen are produced by the corpus luteum. The trigger is low progesterone. 1. Describe the anatomy of an oocyte (egg). Model Answer The secondary oocyte has a haploid pronucleus and is surrounded by the zona pellucida (a layer of clear material), which, in turn, is surrounded by a mass of granulosa cells called cumulus cells. 1. Describe the menstrual cycle in terms of the production and control of production of the female hormone levels including the role of FSH

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and LH in the maturation of oocytes (eggs), production of oestrogen and progesterone and the role of these during the female life cycle. Model Answer 1 Menstruation starts day 1 of the menstrual cycle. Follicles mature during the proliferative stage and produce oestrogen, which stimulates uterine lining thickening and GnRH production. GnRH causes the production of FSH and LH. FSH, LH and oestrogen production are in a positive feedback loop. A peak in LH production stimulates ovulation at about day 14. The corpus luteum produces progesterone and oestrogen which inhibit FSH and LH. During the secretory stage (day 14 28) the uterus prepares for implantation. If fertilization occurs the blastocyst implants at about day 21. If there is no fertilization, the corpus luteum degenerates, progesterone decreases and the lining is shed as menstruation. FSH and LH are produced and the cycle starts again. Model Answer 2 The first day of menses is considered to be day 1 of the menstrual cycle. Ovulation occurs on about day 14. The time between menses ending and ovulation is called the proliferative stage when the follicles mature in the ovary and secrete oestrogen, which causes the endometrium to thicken. Oestrogen secretion stimulates the production of GnRH, which triggers FSH and LH production. FSH stimulates oestrogen so that a positive feedback loop produces increasing surges of FSH and LH. Ovulation occurs in response to the peak in LH. The corpus luteum develops from the ruptured follicle and secretes progesterone and oestrogen. These hormones inhibit GnRH, LH and FSH secretion. The secretory phase is after ovulation and before the next menses. The lining of the uterus is prepared for implantation. If fertilization occurs, the zygote develops into the blastocyst and reaches the uterus 7 to 8 days after ovulation, when implantation occurs. If the secondary oocyte is not fertilized, the corpus luteum decreases the production of progesterone, causing the endometrium to slough away resulting in menses. The decline in progesterone stimulates the production of FSH and the cycle begins again.

6.3.3. Histology of the Ovary

View the animation of Maturation of the Follicle and the Oocyte

Label the diagrammatic representation of the testes.

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6.4.Birth Control and Infertility

List the methods of birth control Behavioural methods Abstinence Coitus interruptus Rhythm method Barrier methods Condom Vaginal condom Diaphragm Spermicidal agents Spermicidal douches Lactation Chemical methods Oral contraceptive/ The Pill RU486 Morning after pill Surgical methods Vasectomy Tubal ligation Abortion

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Non-implantation method

Intrauterine devices / IUD

List the major reasons for infertility. In Malfunction of Female uterine tubes s Reduced hormone secretion Interruption of implantation Endometriosis In Males Low sperm count Abnormal sperm Reduced sperm motility 1. For three popular types of birth control the pill, the diaphragm and coitus interruptus indicate a. the point of blockage of the reproductive process, b. the mode of action, c. the relative efficiency or effectiveness, d. the inherent problems. Model Answer 1 The pill prevents ovulation by decreasing LH and FSH. It is 99% effective. Needs to be taken daily. The diaphragm stops sperm entering the uterus. It is 85% effective. Needs to be available in event of intercourse. Coitus interruptus, the removal of the penis before ejaculation, is unreliable. Sperm maybe present before ejaculation. Model Answer 2 The pill: stops ovulation by reducing LH and FSH release from the anterior pituitary, it is 99% effective, but will be ineffective if not taken daily and can be inactivated by some antibiotics. There is an added risk of heart attack or stroke in females using oral contraceptives who smoke or who have a history of hypertension or coagulation problems. The diaphragm: is a flexible plastic or rubber dome that is placed over the cervix within the vagina and prevents sperm travelling through the cervical canal to the uterus. A spermicide should also be used with it. It is only 85% effective because it can shift. It is reusable, needs to be cleaned and needs to be carried if sex is anticipated. Coitus interruptus: is removal of the penis from the vagina just before ejaculation. It is extremely unreliable because it relies on removal of the penis at the right time and ignores the possibility of sperm being present in preejaculatory fluids emissions.

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6.5. Self Test

Q1. The fluid containing sperm that is produced by the male reproductive structures is a. mucus b. semen c. testosterone d. vestibular fluid Correct. Semen is the viscous fluid containing sperm cells, and secretions of the testes, seminal vesicles, prostate gland and bulbourethral glands. Mucus is a viscous secretion produced by the mucous membranes for protection and lubrication. The correct answer is b. Testosterone is a steroid hormone secreted mainly by the testes. The correct answer is b. The correct answer is b. Q2. During ejaculation the sperm pass through a number of structures. Which of these represents the correct sequence? a. epididymis, vas deferens, urethra b. vas deferens, epididymis, urethra c. epididymis, seminal vesicles, prostate, urethra d. epididymis, vas deferens, seminal vesicles, prostate, urethra Correct. Sperm pass from the testes into the epididymis before entering the vas deferens. The correct answer is a. The sperm do not pass through the seminal vesicles and prostate. These structures add secretions to the urethra. The sperm do not pass through the seminal vesicles and prostate. These structures add secretions to the urethra. Q3. Spermatids a. are diploid cells b. divide mitotically producing spermatogonia c. are haploid cells d. divide meiotically to produce primary spermatocytes Spermatids are haploid cells produced during meiosis. The correct answer is c. Spermatogonia are diploid cells which divide mitotically to produce another spermatogonium and a primary spermatocyte. The correct answer is c. Correct. Spermatids are haploid cells produced when the haploid secondary spermatocyte divides during meiosis II. Spermatids do not divide. They differentiate into spermatozoa. Q4. Primary oocytes a. are formed by mitotic division of oogonia b. are formed by meiotic division of oogonia

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c. are formed by differentiation of oogonia d. differentiate to form an ovum Correct. Oogonia divide by mitosis to produce diploid primary oocytes. Primary oocytes are formed by mitosis of oogonia. The correct answer is a. Primary oocytes are formed by mitosis of oogonia. Primary oocytes divide by meiosis to produce secondary oocytes after meiosis I. Q5. Which of the following produces the male sex hormones? a. Seminal vesicles b. Corpus luteum c. Developing follicles of the testes d. Interstitial cells Correct. The interstitial cells (cells of Leydig) of the testes secrete testosterone. Seminal vesicles produce about 60% of the semen. The correct answer is d. The corpus luteum is produced in the ovary from the ruptured cells of the follicle after ovulation. The testes do not contain follicles. Q6. Which of the following does not add a secretion that makes a major contribution to semen? a. Prostate b. Bulbo-urethral glands c. Testes d. Vas deferens Correct. The vas deferens carries the ejaculate from the epididymis to the ejaculatory duct and on to the urethra. The prostate gland adds prostatic fluid to semen. The correct answer is d. The bulbourethral glands add mucous to the semen. The testes produce the sperm which are ejaculated in the semen. Q7. Which is the correct order for one complete menstrual cycle? a. corpus luteum develops, ovulation, follicle develops b. follicle develops, ovulation, corpus luteum develops c. ovulation, corpus luteum develops, follicle develops d. follicle develops, corpus luteum develops, ovulation The corpus luteum develops after ovulation from the ruptured follicle. The correct answer is b. The follicle develops around the oocyte and is ruptured at ovulation. The corpus luteum develops after ovulation from the ruptured follicle. Q8. A drug that reminds the pituitary to produce gonadotrophins might be useful as a. a contraceptive b. a diuretic

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c. a fertility drug d. an abortion stimulant Gonadotrophins stimulate gonadal growth and function. The correct answer is c. Q9. Which of the following are found in both males and females? a. Erectile tissue b. Vas deferens c. Cervix d. Prostate Correct. Erectile tissue is present in the penis and the clitoris. The vas deferens is present in the male. The correct answer is a. The cervix is present in the female. The prostate gland is present in the male. Q10. The principal male sex hormone is a. oestrogen b. aldosterone c. LH d. testosterone Correct. Testosterone is secreted by the testes and controls the maintenance and development of male reproductive organs and secondary sexual characteristics. Oestrogen is one of the principal female sex hormones. The correct answer is d. Aldosterone is a hormone associated with excretion. LH is produced by both males and females. Q11. The normal site of successful fertilisation that can be expected to result in pregnancy is the a. vagina b. ovary c. uterus d. uterine tube The normal site of successful fertilisation is the uterine tube (Fallopian tube, oviduct). The correct answer is d. The normal site of successful fertilisation is the uterine tube (Fallopian tube, oviduct). The normal site of successful fertilisation is the uterine tube (Fallopian tube, oviduct). Q12. The sac containing the male gonads is the a. testis b. prostate

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c. epididymis d. scrotum The testes are the gonads. The correct answer is d. The prostate is a gland whose secretion contributes to the semen. The epididymis is where the sperm mature. Q13. Female infertility may be due to a. hyposecretion of gonadotropic hormones b. endometriosis c. scarring of the uterine tubes d. all of the above Correct. There are many factors contributing to infertility including infection, lack of sufficient FSH or LH, endometriosis, scarring or even cancers. There are many factors contributing to infertility including infection, lack of sufficient FSH or LH, endometriosis, scarring or even cancers. The correct answer is d. There are many factors contributing to infertility including infection, lack of sufficient FSH or LH, endometriosis, scarring or even cancers. There are many factors contributing to infertility including infection, lack of sufficient FSH or LH, endometriosis, scarring or even cancers. Q14. Describe two important functions of apoptosis. (1 mark) Model Answer Apoptosis (programmed cell death) shapes structures (0.5) in developing offspring and removes abnormal cells (0.5) which could develop into cancer. Q15. Chromosome movement and placement is vital during cell division. How is this done in the cell? What, in general, is a possible consequence if this doesn't function properly? (5 marks) Model Answer 1 Chromosomes are attached by their centromeres (0.5) to spindle fibres (0.5). The spindle fibres contract towards the poles (0.5) dragging the chromatids apart (0.5). If the centromere does not divide (0.5), both chromatids end up in the same new cell (0.5). Total marks: 3/5 Model Answer 2 The spindle brings about chromosomal movement (0.5). Two types of spindle fibres (0.5) are involved. One type extends from the poles to the equator (0.5) which helps the chromosomes move apart (0.5). The chromosomes (0.5) themselves are attached to other spindle fibres that simply extend from their centromeres to the poles (0.5). These fibres get shorter and shorter pulling the chromosomes apart (0.5) as the chromosomes move toward the poles, and eventually disappear (0.5). Failure of this process can lead to non-disjunction (0.5) where both members of a homologous pair of chromosomes go into the same daughter cell (0.5). Q16. Why don't humans look exactly like their parents? In other words, how is each individual assured a different combination of genes than either parent? (2 Marks) Model Answer

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Crossing-over (0.5) recombines the genes on the sister chromatids (0.5) of the homologous pairs. Following meiosis, each gamete has a different combination of chromosomes (0.5). Upon fertilization, recombination of chromosomes occurs (0.5). Q17. Describe how a couple can choose a child who may have or lack certain characteristics such as a genetic disorder. (1 Mark) Model Answer 1 Using in vitro fertilization (0.5), an embryos DNA can be screened for certain genes (0.5), including those causing diseases. Q18. Describe what would happen if large doses of progesterone and oestrogen were given to a woman four days before ovulation. How would this situation differ from the natural hormone and ovarian cycles? (5 Marks) Model Answer 1 Oestrogen and progesterone have a negative feedback effect on FSH and LH (0.5). FSH promotes the development of the follicle and a surge of LH initiates ovulation (0.5). Therefore large amounts of oestrogen and progesterone would prevent ovulation (0.5). Total marks: 1.5/5 Model Answer 2 Administration of a large amount of progesterone and oestrogen, just prior to the luteinising hormone (LH) surge before ovulation (0.5), inhibits the release of Gonadotrophin releasing hormone (GnRH), follicle stimulating hormone (FSH) and LH (0.5), preventing ovulation (0.5). This situation differs from the normal hormone and ovarian cycles because four days before ovulation, oestrogen production is normally low (0.5). It begins to increase as the ovarian follicle matures, until it peaks at ovulation (0.5). This sustained increase in the production of oestrogen by the developing follicles stimulates GnRH secretion by the hypothalamus (0.5), in turn, triggering the secretion of FSH and LH by the anterior pituitary gland (0.5), and thus ovulation (0.5). During this period, the normal progesterone levels are still low and do not begin to rise until after ovulation (0.5), so this has no effect on ovarian cycles before ovulation (0.5). Q19. Describe the secretions of the prostate glands, bulbourethral glands and seminal vesicles and their functions. (6 Marks) Model Answer 1 The prostate gland is located around the urethra below the urinary bladder. It secretes a milky fluid that is discharged into the urethra as part of the semen. Bulbourethral glands are located below the prostate glands and add secretions to semen. Seminal vesicles are attached to the vas deferens near the base of the urinary bladder. They secrete a component of the semen. Total marks: 0/6 Model Answer 2 The prostate gland secretes an alkaline fluid (0.5) to combat the acid of urine (0.5). The bulbourethral glands secrete a buffering (0.5) and lubricating fluid (0.5). The seminal vesicles secrete a fluid that contains energy for the sperm (0.5).

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Total marks: 2.5/6 Model Answer 3 The prostate gland secretes a thin, milky, alkaline fluid that neutralises the acidic secretions of the vagina (1) and it contains proteolytic secretions that break down the coagulated proteins of the seminal vesicles. (1) The bulbourethral glands produce a mucous secretion which lubricates the urethra, helps neutralise the contents of the normally acidic urethra and vagina (1), and provides a small amount of lubrication during intercourse (1). The seminal vesicles secrete a thick, mucous-like secretion containing fructose and other nutrients (1) that provide nourishment to the sperm cells, proteins that weakly coagulate after ejaculation and enzymes that are thought to help destroy abnormal sperm cells (1) Q20. Explain why a penetrating wound to the chest can prevent a lung from inflating. (3 Marks) Model Answer 1 The lungs are surrounded by two membranes separated by a thin layer of fluid. The pressure in this fluid space is lower than atmospheric pressure (0.5). If this space is penetrated, the pressure is equal to the outside pressure (0.5) and the lung collapses. Total marks: 1/3 Model Answer 2 At the end of expiration, the alveolar pressure is equal to the atmospheric pressure (0.5). If the pleural cavity is connected to the outside atmosphere by a penetrating wound, the pressure in the pleural cavity is the same as the atmospheric pressure (0.5). Since the pleural and alveolar pressures are the same (0.5), the alveoli will not expand and the lungs will collapse. Total marks: 1.5/3 Model Answer 3 Each lung is located in a separate pleural cavity that is closed to the outside air (0.5). The contractions of the diaphragm and outer intercostal muscles increase the volume of the pleural cavity (0.5) and decrease the pressure within it and within the enclosed lung (0.5). The pressure in the lung falls below atmospheric pressure and the lung inflates (0.5). If the seal of the pleural cavity to the outside is broken, the expansion of the chest volume fails to decrease the pressure in the pleural cavity (0.5). The pressure in the cavity and the lung remains equal to atmospheric pressure and inflation cannot occur (0.5). Q21. What sensory information is available to the respiratory control centre as it regulates the rate and depth of breathing? (5 Marks) Model Answer 1 The respiratory control centre uses information from chemical receptors in major arteries (0.5) and in the brain (0.5). Special receptors in some arteries check the level of O2. Respiration will increase if the level of O2 decreases (0.5). Receptors near the respiratory control centre check the level of CO2 and the concentration of H+ (0.5). Respiration will increase if these levels rise (0.5). Total marks: 2.5/5 Model Answer 2 The respiratory control centre uses sensory information from chemical receptors in major arteries (0.5) and in the brain (0.5). Receptors in the aorta and internal carotid arteries monitor the partial pressure of oxygen (O2) (0.5). Respiration will increase if the level of O2 decreases (0.5). Receptors in the

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medulla oblongata (0.5) near the respiratory control centre monitor the partial pressure of carbon dioxide (CO2) and the concentration of H+ (0.5). Respiration will increase if these levels rise (0.5). Total marks: 3.5/5 Model Answer 3 The respiratory control centre is in the medulla oblongata. An increase in carbon dioxide (CO2) causes changes in the blood pH (0.5) and the concentration of H+ ions (0.5). These changes are monitored by receptors near the control centre (0.5) which is directly stimulated to increase the respiration rate (0.5). Chemoreceptors in the carotid and aortic bodies (0.5) monitor the concentration of oxygen (O2) in the blood (0.5). These bodies interact with the respiratory control centre to increase the rate and depth of breathing (0.5) when O2 concentration falls (0.5). However O2 concentration is not as important as CO2 in regulating breathing. The respiratory control centre sends nerve impulses to the diaphragm and intercostal muscles to contract, expanding the thoracic cavity so air enters the lungs (0.5). When the impulses stop, the muscles relax and the air is pushed out (0.5).

7.DEVELOPMENT
7.1.Objectives
In the Development lectures we will look at the stages of development from fertilisation through to birth and then the stages of aging from birth to death. HBOnline 6 Development section will cover the major features of development, as well as considering birth defects and sexually transmitted diseases. Both of the latter have significant implications in the world today.

Objectives
After completing the lectures, HBOnline 6 and Unit 6 practical on development you should be able to recognise the basic features of fertilisation and cleavage describe the processes of gastrulation, neurulation and morphogenesis describe the major events in human development. explain the terms congenital abnormality and teratogen and give some example. list the four major cellular processes underlying embryonic development define and describe cleavage and gastrulation explain what is meant by organogenesis describe the implantation of the human embryo explain the functions of the yolk sac, amnion, chorion and allantois in human development describe the role of the human placenta in embryonic nutrition, excretion, respiration and defence understand the hormonal interactions between mother and embryo during human pregnancy explain what is meant by "artificial culture of mammalian embryos to the blastocyst stage" discuss embryo transfer in humans give the timetable of major events in the development of the human embryo and foetus.

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explain how the development of sexual differentiation takes place state the three major steps in the birth process describe the mechanism that initiates human birth explain how the foetus at birth changes from placental respiration to lung breathing describe the range of birth defects identified today document ways of preventing birth defects.

Activities
The following list contains all activities covered on this topic. Those activities marked with an asterisk (*) will be done in the timetabled Development/Inheritance practical. Overview of Reproduction and Development Major Stages of Development o Fertilisation to Foetus Pregnancy Testing* Birth Defects Congenital Malformation* Development Websites Development Card Game Self-test

7.2.Overview of Reproduction and Development

You can view the video on Reproduction at the Lizard Lounge after borrowing it from the FYB Office which can be found in the Carslaw Building, Level 5, Room 519.

7.3.Major Stages of Development

Read
Seeley's Essentials of Anatomy and Physiology (7th ed.) Chapter 20 - Development, Hereditary, and Aging Prenatal Development

the Human fertilization, cleavage and implantation in the uterus

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1. Uterus 2. Myometriu m 3. Endometriu m 4. Ovulated oocyte 5. Follicle cells 6. Zygote 7. Two cell stage 8. Four cell stage 9. Morula 10. Blastocyst

11. Inner cell mass 12. Trophoblast 13. Implanted embryo 14. Trophoblast forming placenta 15. Ovum 16. Implantation 17. Fertilisation 18. Ovary 19. Oviduct (fallopian or uterine tube)

about fertilisation and cleavage.

At ovulation the secondary oocyte is released from the ovary. Fertilisation results in the completion of the meiotic division to form an ovum with a pronucleus which then fuses with the sperm pronucleus. The first mitotic divisions of the fertilised egg are called cleavage. During the rapid increase in cell number, the overall size of the organism remains constant and is equalto that of the fertilised egg. At each successive cell division, the daughter cells arereduced in size. Large quantities of nutrients are required for the numerous cleavage divisions. These nutrients come mostly from the yolk contained within the cytoplasm of the ovum. The fluids from the oviduct/ fallopian tube also help nourish the zygote. 1. Define cleavage. Model Answer

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Successive mitotic divisions of the fertilised ovum, which ends with the formation of the blastula. 2. State very briefly what happens in development in the second week after fertilisation. Model Answer Implantation of the blastocyst. Formation of the bilayer embryo and differentiation of the primary germ layers. Formation of three chambers around the embryo, three layers of extraembryonic membranes, and formation of the early placenta. 3. What are the four functions of the placenta? Model Answer 1 1. Transfer of oxygen, carbon dioxide, nutrients and wastes from foetus to mother. 2. Produces enzymes to break down harmful molecules and prevent them reaching the foetus. 3. Secretes HCG. 4. Forms a barrier between the two blood circulations. Model Answer 2 1 - Transfer. Transfer of nutrients and oxygen from the maternal blood vessels to the foetus (via the umbilical vein) and transfer of CO2 and wastes from the foetus (via the umbilical arteries) to the maternal blood vessels. 2 - Metabolism. Synthesis of a great variety of enzymes which are capable of converting biologically active molecules e.g. hormones and drugs into less active water soluble molecules. The placenta thus prevents potentially dangerous molecules from harming the foetus. 3 - Endocrine secretion. Secretes HCG from start to maintain pregnancy. The placenta becomes the major sex-steroid gland from 5.5 weeks after the start of the pregnancy. It also secretes hormones which are similar to some produced by the anterior pituitary gland. 4 - Barrier to transfer of molecules from maternal -> foetal circulation -> placental barrier. Read Seeley's Essentials of Anatomy and Physiology (7th ed.) Chapter 20 - Development, Hereditary, and Aging Formation of the germ layers Neural tube and neural crest formation Formation of the general body structure Development of the organ systems

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the stages of foetal development

Label: amnion, amniotic cavity, chorion, chorionic villus, extraembryonic coelom, foetus, placenta, umbilical cord

1. Foetus 2. Amniotic cavity 3. Amnion 4.Extraembryonic coelom

1. 2. 3. 4. 5.

Chorionic villus Amniotic cavity Foetus Placenta Umbilical cord

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7.4.Pregnancy Testing
This activity will be covered in the Development/Inheritance practical as timetabled.

7.5.Birth Defects
Read Seeley et al. Chapter 20 - Development, Hereditary, and Aging The newborn Clinical Focus: Disorders of Pregnancy and Birth
1. Describe at least three ways of detecting birth defects and discuss the advantages and disadvantages of the methods for the mother and the developing foetus.

Model Answer
Amniocentesis: done at 15th to 17th weeks, then takes maybe 4 weeks for the test to be completed; it is invasive and so carries some risk to mother and foetus. Because it is done so late in the pregnancy, there is little time to terminate the pregnancy if a defect is detected. Chorionic villi sampling: done at 5th to 12th week and tests are completed immediately, it is invasive for mother and poses a greater threat of the unborn child than amniocentesis. For example, increased foetal risk of finger and toe defects is linked to CVS. Screening of eggs: eggs from the ovary are removed and the first polar body is tested. If the mother is heterozygous and if the polar body has the gene for the defect, the egg itself will not carry the defective gene and this egg is implanted by IVF. Ultrasound: done at about 10 weeks, non-invasive, but does not identify many genetic defects they need to be defects in external structures so that the sound waves involved in producing the image can show the problem. Foetal heart sounds can be detected with an ultrasound stethoscope by the 10th week and with a conventional stethoscope by the 20th week. Noninvasive, but gives limited information.

7.6.Congenital Malformation

1. Knowing the adverse effects of various substances on the developing foetus, discuss ways of controlling their use by pregnant women. Model Answer Some prescription drugs are banned to everyone, others are prescribed only to non-pregnant women (teratogens), pregnant women are encouraged to abstain from smoking, alcohol intake (foetal alcohol syndrome), and drug use (cocaine addiction). 2. Why are embryos sensitive to teratogens during critical periods in development? Model Answer The critical periods of development of a particular organ are when the most rapid cell divisions are occurring. The critical period varies in accordance with the timing and duration of the period of increasing cell numbers for the tissue or organ concerned.

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7.7.Development Card Game

Spermatogenesis

Oogenisis; Right order

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Fertilisation; Right order

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Right order of implantation

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Orgagenosis; in right order

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Growth and Development

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7.8. Development Websites

Access the following web sites to review the development of the embryo and foetus 1. http://www.med.upenn.edu/meded/public/berp - subsection Overview University of Pennsylvania Health System 2. http://visembryo.com 1. University of Pennsylvania Health System http://www.med.upenn.edu/meded/public/berp - subsection Overview

Basic Embryology Review Program

Embryonic Disc Nervous System Heart Eye Ear Branchial Arches Face Gastrointestinal Urogenital Limbs

8.INHERITANCE
8.1.Objectives
INHERITANCE, DNA AND FRONTIERS OF HUMAN GENETICS
The genes we inherited from our parents control our anatomical and physiological features and even some of our behavioural characteristics. They are in turn passed to the next generation. How these genes are passed on and the rules governing their expression are explained in this section.

OBJECTIVES
After completing the lectures, HBOnline 6 and Unit 6 practical on inheritance, DNA and frontiers of human genetics you should be able to understand the basics of amplifying DNA via the Polymerase Chain Reaction (PCR) describe how errors during meiosis lead to abnormalities use the software NIH Image to analyse banding patterns on chromosomes use and interpret human pedigrees PCR cell culture transgenic animals gene therapy differences between in vivo and ex vivo techniques draw and interpret human pedigrees describe some simple chromosomal abnormalities understand the methods and purpose of pre-natal testing understand the basis of genetic counselling understand the strategies used for mapping human disease genes, linkage, in situ hybridisation and somatic cell genetics

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understand how DNA evidence can be used to exclude crime suspects understand the pros and cons of gene sequence and gene product patents understand the ethical and psychological difficulties associated with genetic testing appreciate issues related to genetic testing and health and life insurance understand the goals and rationale for the Human Genome Project appreciate that the science of bioinformatics develops strategies for efficient storage and retrieval of DNA sequence information.

ACTIVITIES
The following list contains all activities covered on this topic. Those activities marked with an asterisk (*) will be done in the timetabled Development/Inheritance practical. Human Chromosoomes o Autosomal and Sex Chromosomes o Karyotypes and Nondisjunction o Chromosome Banding Pedigrees o Interpretation of Human Pedigrees A Virtual Molecular Genetics Lab Human Genome Project Consideration of Genetic and Ethical Issues Self-test

8.2.Pedigrees
Read
Seeley's Essentials of Anatomy and Physiology (7th ed.) Chapter 20 - Development, Hereditary, and Aging Genetics The word 'pedigree' can refer to the pattern of transmission of a trait in a family tree, the family tree itself, or the mere fact that the family tree is known (as in pedigree cattle). We will use it to mean the family tree. PEDIGREE SYMBOLISM Male Female Mating between unrelated individuals Consanguineous mating (i.e. between related individuals) Siblings (or sibs) in birth order from left to right Individuals showing the trait under consideration Individuals not affected by the trait under consideration

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Individuals carrying the trait under consideration An Example of a Pedigree

The Roman numerals indicate the generations from top to bottom; the Arabic numerals indicate the individuals within a generation from left to right (though numbers are not usually written on the pedigree). Together, these two numbers allow each individual to be identified. Parents are joined by a mating line. Offspring of a single mating form a sibship and the children are referred to as sibs (short for siblings, i.e. brothers and sisters). Family refers to both parents and their offspring. Kindredrefers to a broader set of related individuals. Text Which individual is III.6s paternal grandfather? I.1 Which individuals possess the trait indicated by a filled symbol? II.3 and II.4 What is the relationship between individuals III.5 and I.3? III.5 is the grandchild, I.3 is her maternal grandfather INTERPRETATION OF HUMAN PEDIGREES The study of the way traits are inherited is conducted often to determine the nature of genetic disorders. Understanding the nature of transmission provides information about where genes are located within the genome and whether genes are abnormally abundant, missing or defective. The inheritance of traits within a family can be followed through the generations using a human pedigree (family tree). From examining the mode of gene transmission from generation to generation, it is possible to determine the nature of genes, i.e.whether sex-linked or autosomal, dominant or recessive. Here are some simple rules that assist in the interpretation of pedigrees. These general rules are also explained in your textbook.

POINTS TO REMEMBER IN DETERMINING THE GENETICS OF A TRAIT Autosomal Dominant Traits

If a trait is dominant, it will normally appear in every generation back to the time when the original mutation occurred. For example, if a child shows the trait, then at least one of his or her parents will show the trait also. Heterozygotes are affected i.e. AA and Aa show trait, aa does not. Males and females are equally affected.

Autosomal Recessive Traits

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If a trait is recessive, it may not appear in a particular generation or several generations because it is masked by the dominant phenotype. For a child (whose parents do not show the trait) to show that characteristic, both parents must beheterozygous. Parents Bb x Bb gametes (B) (b) (B) (b) offspring BB Bb Bb bb When both parents show the trait, all their children should show it. Parents dd x dd gametes (d) (d) offspring all dd

Sex-linked Recessive Traits

If a trait is sex-linked recessive, more males than females will show the trait. For a girl to show the trait, her father must show it too. N.B. Fathers do not pass on sex-linked (X chromosomal) traits to their sons. An affected female passes the trait to all her sons. In assessing a pedigree for the likely mode of inheritance of a rare monogenic trait (i.e. a trait caused by a single gene), the following examples should enable you to determine some of the possible modes of inheritance.

Text entry exercise

can you decide which mode of inheritance is the most likely (dominant, recessive, sex-linked, autosomal) or do you need more information? If all affected individuals have at least one parent similarly affected (i.e. it is seen in each generation), it is most likely to be dominant. If all affected individuals do not have at least one parent similarly affected (i.e. it is not seen in each generation), it is most likely to be recessive. If there are roughly equal numbers of males and females among affected individuals, it is probably autosomal. If there are not roughly equal numbers of males and females among affected individuals and the trait is recessive with mostly males affected then it is probably X-linked. Affected females must have an affected father. If only males affected it could be sex-limited e.g. baldness. If the trait is dominant and all daughters of an affected father have it but not the sons, then it is probably X-linked. If the trait is dominant and half the daughters, half the sons and the mother are affected then it is probably also X-linked. If the parents of affected individuals are related to each other, this often brings to lightrecessive traits that would not otherwise show up. If the parents of affected individuals are not related to each other there is not enough information to make a decision. Need to look for more clues by investigating if more distantly related members of each family are affected. If affected fathers pass the trait to their sons, X-linkage is not a possibility. If affected fathers do not pass the trait to their sons, it is probably X-linked. Look at the pedigrees below and fill in the blanks. PEDIGREE 1

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The shaded symbols represent individuals who have a rare trait caused by a dominant gene A.

Is the gene autosomal or X-linked? Autosomal Reason(s) for the decision. Trait passed from father (II.2) to son (III.2), which cannot happen with X linked. What proportion of the progeny of III.I and III.2 would show the trait? 0.5 What proportion of the progeny of III.6 and III.7 would show the trait? 0.75 PEDIGREE 2 The shaded symbols represent individuals who have a rare trait caused by a recessive gene.

Is the gene autosomal or X-linked? Autosomal. Reason(s) for the decision. II.2 and II.3 must be heterozygotes to have offspring showing the trait. If it was X-linked fathers would not be able to pass the trait on to sons.

8.3.A Virtual Molecular Genetics Lab

Your mission is to go to the Virtual Bacterial ID Lab which is part of the Howard Hughs Medical Institute Biointeractive site athttp://www.hhmi.org/biointeractive/ (click on "Virtual Labs", scroll down to "The Bacterial ID Lab" and click on the image to enter the program), and learn how to isolate and amplify DNA using the polymerase chain reaction (PCR). . There is a link in the Computer-Assisted Learning Modules section of the Virtual Learning Environment. Do the first 2 activities of the Lab. They are: 1. Sample Preparation and 2. PCR Amplification, of course you can go on with more activities if you wish!

Short answer exercise

1. After adding your bacterial colony to the micro centrifuge tube, why did you add digestive enzymes (digestive buffer) to the tube and leave it for a few (virtual) hours?

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Model Answer The digestive buffer contains proteolytic enzymes that eat the cell wall. 2. How do you get rid of cell debris (bits of broken membrane, mitochondria etc) from your sample digest? Model Answer The cellular debris is spun down in the centrifuge and appears as a solid deposit (pellet) at the bottom of the tube. 3. You took a tube out of a centrifuge and removed some of the supernatant with a pipette. What is meant by supernatant? Model Answer The supernatant is the liquid that overlies the pellet. In this sample the supernatant contains the DNA.

PCR Amplification
1. What is the main purpose of PCR amplification? Model Answer Polymerase Chain Reaction is a technique that allows many copies of the DNA to be made. 2. The PCR master mix contains water; a buffer to keep the mixture at the correct pH for the PCR reaction and what else? Model Answer Large quantities of the four nucleotides (adenine, guanine, cytosine and thymine) and the primers that bind the 16S rDNA region to initiate the replication process. It also contains heat-stable DNA polymerase that extends the copy DNA strand. 3. What is the effect of "melting" heating to 95 degrees for 30 seconds on DNA structure? Model Answer Separates the two DNA chains in the double helix. 4. What is meant by "annealing"? Model Answer The binding of the primer to the single-stranded DNA. 5. What happens in the extension phase of PCR and what enzyme is needed for extension to take place? Model Answer DNA polymerase is used to extend the copy DNA strand. 6. Imagine you are a forensic scientist. You have a piece of hair from a crime scene and a suspect has been arrested. You are going to use DNA evidence to match the DNA from the hair to that of the suspect. The hair contains one piece of DNA, you need 500,000 DNA copies minimum for the analysis, what would you do? Model Answer 1. DNA extraction a. Using a proteolytic enzyme to break down the cell membrane of cells. b. Heat sample to deactivate the enzyme. c. Centrifuge and remove supernatant containing DNA. 2. PCR amplification a. Add PCR master mix to supernatant (containing primer for wanted DNA sequence).

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b. Place in thermocycler (PCR machine) and allow to go through 3 steps: Melt, Anneal and Extend several times (approx. 30). 3. PCR purification: Remove PCR master mix and collect amplified DNA. 4. Sequence DNA and analyse. 7. How could you as a scientist use the PCR technique? Model Answer Amplify DNA to use for forensic purposes, to identify if a mutation exists in a gene using primers for a particular region of DNA, to make complementary DNA from mRNA. 8. Describe the three major steps in each PCR cycle. Model Answer Separation of the two strands of DNA, Annealing of the primers to the single stranded DNA to allow extension, Extension of the new strand with the addition of dNTPs.

8.4.Self Test
Q1.

Could the character represented by the solid symbols in the above pedigree be explained on the basis of a a. dominant character due to an autosomal gene? Incorrect. If the gene is dominant, at least one parent of III 1 should also show the trait. The correct answer is b. b. recessive character due to an autosomal gene? Correct. If both parents of III 1 are heterozygous, then III 1 will show the trait. c. dominant character due to a sex-linked gene? Incorrect. Males do not pass sex-linked genes to their sons. d. recessive character due to a sex-linked gene? Incorrect. Males do not pass sex-linked genes to their sons. Q2.

In the above pedigree, could the trait indicated by the solid symbols be recessive and due to a sex-linked gene? Explain your reason. No. III.1 shows the trait, but her father does not. So the trait cannot be sex linked but could be autosomal dominant or recessive. More information is required.

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Q3. What are the 3 main steps in the Polymerase Chain Reaction (PCR). 1. Heating to separate strands. 2. Hybridisation of primers. 3. DNA synthesis from primers. Q4. What is an RFLP analysis? RFLP stands for restriction fragment length polymorphism, RFLP looks at the different fragment lengths produced when a restriction enzyme cuts at specific DNA sites. Because different DNA will have the specific cutting sites at different points on the strand the enzyme will produce different length segments from different DNA molecules. These are detected by electrophoresis and seen as bands. Q5. Name two uses of RFLP analysis. 1. Paternity testing. 2. Forensic analysis. Q6. How are the bands produced with RFLP detected? They are usually stained with compounds such as Azure A, or ethidium bromide that can be seen under UV light. Q7. Why is PCR often performed on DNA samples before RFLP analysis? RFLP analysis needs a certain amount of DNA so that the band staining becomes dark enough to see, PCR can amplify a tiny amount of DNA that would not otherwise be detected. Q8. If you start with two strands of DNA and you know the number of strands needed for a RFLP analysis, what simple mathematical function can you do to determine the number of cycles of PCR you will need to perform to stain the bands adequately? Explain your answer. The square root of the final number of strands will give you the number of PCR cycles. Because the PCR reaction doubles the strands at each cycle if you start with 2 strands you will have 2 to the power of the number of PCR cycles that you perform. In other words if x is the number of PCR cycles, the final number of strands is 2x. If you take the square root of the final number of strands it will equal x. Q9. Imagine you are a forensic scientist and you have samples from a crime scene containing miniscule amounts of DNA that you are planning to use for RFLP analysis, why would you be very careful to make sure your samples do not become contaminated with bacteria? The samples would need to be amplified with PCR. If bacterial DNA is present, this will also be amplified and will lead to artifact band products that will obscure the results of your analysis.

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