FACULTY OF APPLIED SCIENCES AND COMPUTING

BABS2213 PRINCIPLES OF GENETICS

PRACTICAL 6

TITLE: THE SEX CHECK

NAME: CHOONG MEL JUNE

GROUP: RBS 2 A1

STUDENT ID: 14WAR10521

DATE: 18 JUNE 2014

DEMONSTRATER: DR LOH KHYE ER

Objectives:To perform buccal smear test for investigation of the existence of Barr’s body.

Introduction:Sex chromosome, a pair of chromosomes is using to determine whether an individual

is male or female. The sex chromosomes of human beings are categorized as X and Y. In humans, the sex chromosomes comprise one pair of the total of 23 pairs of chromosomes; meanwhile other 22 pairs of chromosomes are called autosomes.

Individuals that having two X chromosomes (XX) are female; while individuals that having one X chromosome and one Y chromosome (XY) are male. Comparing X chromosome and Y chromosome, X chromosome has a large autosomal chromosome with a long and short arm, while Y chromosome has one long arm and a very short second arm. This path to maleness or femaleness originates at the moment of meiosis when cell division to produce gametes, or sex cells having half the normal number of chromosomes. In meiosis stage, the male XY sex-chromosome pair separates and passes on X or Y to separate gametes. This result one-half of the gametes that are formed contains the X chromosome and the other half contains the Y chromosome. The female has two X chromosomes, and all female egg cells normally carry a single X. The eggs fertilized by X-bearing sperm become females (XX) and fertilized by Y-bearing sperm become males (XY).

Each family member normally carries alleles of the same genes unlike the paired autosomes. The paired sex chromosomes do not carry an identical complement of genetic information. The X chromosome, being larger, carries many more genes than does the Y. Traits controlled by genes found only on the X chromosome are said to be sex-linked. Recessive sex-linked traits occur far more frequently in men than in women. This is because the male who inherits the recessive allele on his X chromosome has no allele on his Y chromosome to counteract its effects. On the other way, in order to fully display the trait, the female must inherit the recessive allele on both of her X chromosome. A woman who inherits the recessive allele for a sex-linked disorder on one of her X chromosomes may show a limited expression of the trait. This is due to one of the X chromosomes is randomly deactivated in each somatic cell of a normal female. Deactivation of X chromosome can be seen as a small, dark-staining structure, which known as the Barr body in the cell nucleus.

Barr bodies can be defined s a small dark stained mass of X chromosome, which is an inactive X chromosome in a female’s somatic cell. It is present in the nuclei of all cells except the germ cells. It is also called as sex chromatin as it indicates the presence of sex hormone. During interphase, any cell with more than one X chromosome will exhibit (n – 1) Barr bodies, which can be said as each excess of X chromosome will form one Barr body. This means that 46, XX women will have cells that exhibit one Barr body, while the cells from a 46, XY male will not exhibit any Barr bodies.

Besides, Barr bodies are present in other parts of body cells too. For example, skin cells, epithelium cells and nerve cells. In a normal female, the number of Barr bodies present is comparatively less than the total number of X-chromosomes.  Barr bodies are present in the

nucleus and if the Barr body is present in the nucleus of the cell, it is referred to as positive. If they are present in other cell apart from nucleus cell, it is referred to as negative.

In 1961, Mary Lyon proposed that dosage compensation in mammals occurs by the inactivation of a single X chromosome in females, which a highly condensed structure in the interphase nuclei of somatic cells in female that was not found in male, which known as the Barr body. On the other way, it can be explained that the Barr body is a highly condensed X chromosome. This compact appearance reflects its inactivity. Hence, there is only one functioning copy of each X-linked gene in the cells of female.

In X-inactivation, female embryos randomly inactivate one X chromosome in each cell. X-inactivation requires a locus on the X, called the X-inactivation center. At this locus, inactivation occurs in response to a developmental cue, which is present only at specific stages of embryo development. Inactivation occurs because of a specific type of RNA, which binds to one X chromosome, preventing transcription of the genes on this particular copy. In addition, enzymes add methyl groups to the DNA of the inactive X, resulting in repression of transcription. The inactivated X is visible during interphase in mitosis. It replicates in the S (synthesis) phase of the cell cycle later than does the active copy. Inactivation of one of the two X copies in a female leaves only one active X chromosome in any cell. An individual who has three X chromosomes has two inactivated copies of the X, producing two Barr bodies. Because the X is inactivated randomly in cells, one cell could have the maternal X inactivated, while the adjacent cell could have the paternal X inactivated. This causes a pattern of gene expression called mosaicism, which occurs when different alleles of X-linked genes are expressed in different cells.

A short region on the X chromosome called the X-inactivation center (Xic) is known to play a critical role. The counting of human X chromosomes is accomplished by counting the number of Xics. A Xic must be found on an X chromosome for inactivation to occur. If one of the two X chromosomes in a female is missing its Xic due to a chromosome mutation, a cell counts only one Xic and X inactivation does not occur. Having two active X chromosomes is a lethal condition for a human female embryo.

The expression of a specific gene within the Xic is required for the compaction of the X chromosome into a Barr body. It is named Xist (for X-inactive specific transcript). The Xist gene on the inactivatedX chromosome is active, which is unusual because most other genes on the inactivated X chromosome are silenced. The Xist gene product is an RNA molecule that does not encode a protein. Instead, the role of the Xist RNA is to coat the X chromosome and inactivate it. After coating, other proteins associate with the Xist RNA and promote chromosomal compaction into a Barr body.

The mechanism of X inactivation is also known as the Lyon hypothesis. The Lyon hypothesis states that during early development, about the 100 cell stage in humans, one of the X chromosomes in a female gets turned off and this is maintained in all descendant cells of the clone. Which of the two X chromosomes gets turned off in each of the 100 cells is purely a random event except where one of the X chromosomes is abnormal (deletion, insertion, inversion, etc.) An abnormal X is always turned off. However, if there is a translocation between

an X chromosome and an autosome, the normal X is turned off and the translocation X remains active.

There are some sex chromosome abnormalities. One of it is turner syndrome. It is the leading cause of autosomal chromosome abnormalities, so is non-disjunction the leading cause of the X and Y abnormalities. In autosomal abnormalities an increase in non-disjunction was associated with increasing maternal age. In sex chromosome abnormalities, one additional source of non-disjunction can be identified, the problem of X and Y pairing at first meiotic division in spermatogenesis. The X and Y have homology only in a small region (called the pseudo-autosomal region) which lies near one end of each chromosome. Pairing occur only in this small region rather than pair along their entire length. At first meiotic division in the male, pairing of X and Y looks more like end to end pairing than longitudinal pairing. This undoubtably adds to the frequency of non-disjunction. Turner syndrome individuals can also result from early mitotic non-disjunction, being mosaic 46, XX/45, X. The phenotype of Turner syndrome individuals differs significantly from the normal female, even though the normal female has only one functioning X chromosome in each cell. The events of embryogenesis during the time both X chromosomes are functioning in the female must be critical, as well as those few regions of the X chromosome that are not inactivated.

There are some non-meiotic chromosomal abnormalities too. One of it is inversions. Inversions involve two chromosomal breaks and rejoining, with the broken piece reincorporated in the opposite orientation from which it naturally occurs. When they include the centromere, they are called pericentric inversions. When they do not include the centromere, they are called paracentric inversions. Both types of inversions arise in mitotic cells. If they arise in precursors of the gametes, they may produce abnormal genomes as they progress through meiosis. Recombination between homologous chromosomes is a necessary part of every normal meiosis. The probability for non-disjunction is greatly increased if there is no recombination. However, recombination between a chromosome with an inversion and its normal homolog may result in two abnormal chromosomes being produced. The results of a paracentric inversion going through meiosis, with a recombination within the inversion, are shown in Figure 10. Of the four gametic products, one is normal, one has the inversion, one has an acentric chromosome, and one has a dicentric chromosome. The acentric chromosome cannot survive. The dicentric

chromosome may be pulled apart during mitosis, with a random loss or gain of genetic material.

Paracentric InversionThe results of a pericentric inversion going through meiosis, with a recombination within the inversion are shown in below. Again, four different gametic products are produced, one normal,

one has the inversion, and two have duplicated portions and deleted portions. The effect on the phenotype is almost always deleterious, but the magnitude of the effect depends upon the size of the duplications and deletions, and where they occur.

Pericentric Inversion

Materials:Cheek cell, microscopic slide, cover slip, toothpick, thionin, distilled water, 50% ethyl alcohol, 70% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol, 6N Hydrochloric acid, xylene and microscope, immersion oil.

Methodology:A. Obtaining the Smear

Mouth was rinsed several times with tap water.

The lining of the cheek was scrapped with toothpick and smeared it on a clean slide.

The smear was placed to air dry for 30 seconds.

B. Staining and Mounting The slide was placed in 95% ethyl alcohol, 70% ethyl alcohol, 50% ethyl alcohol and

distilled water for 2 minutes according the sequences.

The slide is placed in 6N Hydrochloric acid for 10 seconds in precise.

The slide was transferred to distilled water for 10 to 15 seconds.

The slide was placed in the 1% aqueous thionin for 10 to 15 minutes.

The slide was placed in 50% ethyl alcohol, 70% ethyl alcohol 95% ethyl alcohol and absolute ethyl alcohol for 30 seconds according the sequences for dehydration purposes.

The slide was transferred into xylene for 1 minute for cleaning purpose.

The slide was covered with a cover slip and addition of oil added and observed under the microscope.

The male and female cells were observed.

50 cells of female were examined and counted the cell that exhibits sex chromatin in percentage.

Results: (refer the microscopic drawing A and B)- Buccal smear of female shows one dark stained Barr bodies appear as a circular, disk

shaped, which are attached to nuclear membrane within the cell. (microscopic drawing A)- Buccal smear of male shows nuclear membrane within the cell, which represents the

absence of Barr bodies. (microscopic drawing B)

Discussion:A biological test was performed to identify the sex determinants with the help of these

Barr bodies. In this test of buccal smear, Barr bodies appear as a circular, disk shaped attached to their nuclear membrane in female cheek cell. They play a vital role in representing the presence of   inactive X-chromosome, which are present in the vegetative or the somatic cells.

In female cells, Barr bodies or sex chromatin can be observed in 4 positions:

They will be attached to the nucleus in nerve cells. They will be attached to the nuclear membrane. They are found freely in the nucleoplasm. They can also be found in nuclear expansions.

Barr body testing is a test to determine the presence of multiple X chromosomes in a cell. When more than one X chromosome is present in a cell, only one X is allowed to remain active, and any excess X chromosomes are randomly inactivated and condensed. The condensed, inactivated X chromosome is called a Barr body. In this method of testing, cells are frozen in interphase, when the Barr body is condensed, and stained to allow viewing under a microscope.

There are pros and cons when we using Barr body testing. The advantages of Barr body testing are it was much more objective, and less demeaning to women, as only a blood sample was needed to obtain a cell sample that could be tested, instead of forcing women to strip off their clothes and undergo a physical examination. Besides, another advantage of Barr body testing is that there is a much greater potential for protecting the privacy of the athletes as compared to the previous method of using nude parades.

Apart from advantages, there are disadvantages for Barr body testing too. Barr body gender verification testing is based on the assumption that all women have two X chromosomes and therefore display one Barr body. Meanwhile men, who have one X and one Y chromosome, display no Barr bodies. Furthermore, testing is based on the assumption that every cell in an individual contains the same genetic material. First error may be occurred is Barr body testing incorrectly excludes some women from competing who do not have an advantage. Women with Turner’s syndrome (45, XO) will not have a Barr body, so they will not pass a Barr body test. However, they are considered female and the syndrome does not confer a competitive athletic advantage. Moreover, Barr body testing can categorize the same individual as a man or a woman.

Some individuals have mosaicism that some of their cells are XX while others are XY. Depending on the proportion of the cells that are XX, the individual may appear completely phenotypically female. This causes the incorrectness in Barr body testing. However, depending on the cells taken, the same individual can be classified as either a man or a woman at different instances.The process of X inactivation can be divided into three phases:

Phase 1: InitiationDuring initiation, it occurs during embryonic development. One of the X chromosomes remains active, and the other is inactivated.

Phase 2: SpreadingDuring the spreading phase, the chosen X chromosome is inactivated. This spreading requires the expression of the Xist gene. The Xist RNA coats the inactivated X chromosome and recruits proteins that promote compaction. The spreading phase is so named because inactivation begins near the Xic and spreads in both directions along the X chromosome.

Phase 3: MaintenanceOnce the initiation and spreading phases occur for a given X chromosome, the inactivated X chromosome is maintained as a Barr body during future cell divisions. When a cell divides, the Barr body is replicated, and both copies remain compacted. This maintenance phase continues from the embryonic stage through adulthood.

The sex chromatin is not due to XX chromosomes but is due to the fact that one X chromosome is tightly coiled along most or all of its length during interphase. As it is tightly coiled so it become visible when stained. The sex chromatin is not present in the nuclei of males as though they also have one X and Y chromosome because X in the males does not coil but remains uncoiled (extended) in interphase nuclei. This extended chromosome gives information that both X and Y chromosome are derived from autosomes but during evolution Y chromosome has lost most or all of the genes which are not concerned with the determination of sex. The X chromosome has retained about 50 genes which have nothing to do with sex. If these genes which control the information of enzymes do not work, the body cells of male would suffer from many defects. So an active extended X chromosome is essential both in the body cells of males and females. One of the X chromosome remain coiled in female and other remains extended because if both X chromosome are in extended form then enzyme production will be excessive which can cause serious effects.

From this experiment, 50 squamous epithelial cells from a human female were examined. There are 30 out of 50 exhibits sex chromatin. Hence, the percentages of these 50 cells reveal sex chromatin is

3050

×100 %=60 %

It reported high percentage of sex chromatin presence in this human female. Hence, we concluded it is healthy cells which in the range of 50% and 65% demonstrate sex chromatin.

Meanwhile, the incidence of sex chromatin among 50 oral mucosal cells obtained from a human male is none. As the statistical incidence of sex chromatin in male cells normally ranges from about 2% in oral smears, but form this experiment, we couldn’t obtained any of it.

As observed from the cells of the human female, there is no one which has more than one sex chromatin body. This can be explained that a typical human female has only one Barr body per somatic cell. If XXX individuals have two Barr bodies, she is categorized under XXX syndrome. It is a form of chromosomal variation characterized by the presence of an extra X chromosome in each cell of a human female, which also known as Triple X syndrome. Triple X syndrome is not inherited, but usually occurs as an event during the formation of reproductive cells (ovum and sperm). An error in cell division called non-disjunction can result in reproductive cells with additional chromosomes.

Conclusion:From buccal smear test, only epithelial cells from human female exists Barr’s body. There are 30 out of 50 squamous epithelial cells from a human female exhibits sex chromatin. The percentages of these 50 cells reveal sex chromatin is 60%. Meanwhile, the incidence of sex chromatin among 50 oral mucosal cells obtained from a human male is none.

References:1. Ritchie, R., J. Reynard, and T. Lewis. “Intersex and the Olympic Games.” Journal of the

Royal Society of Medicine 101.8 (2008): 395-99. Print.2. Rougeulle, C., Avner, P. (2003), Controlling X-inactivation in mammals: what does the

centre hold?. j.semcdb 14, 331-340. (Abstract)3. Alberts,B., Johnson,A., Lewis,J., Raff,M., Roberts,K., Walter,P., (2002), Molecular

Biology of the Cell, Fourth Edition, (428-429) Garland Science, 0-8153-4072-9 (Web Edition, Free access)