The aim of this practical class was to provide students with the understanding of basic methods used in the amplification of specific loci whose role is to provide information about particular chromosomes (in this case, the sex chromosomes). PCR was used in order to amplify each locus and some of the DNA samples used were of known and unknown donors. The sex was derived from the assays of each unknown sample. Finally, students were asked to consider the ways that the clarification of the results of DNA based sex testing can be affected due to mutations and rearrangements on chromosomes.
By examining the students' data and the AMELX/AMELY locus data the expected results were found to be present. More clearly, the zero samples (samples numbered as 3 for each locus) have given no results as they were used as controls. The expected results were also given from the known normal male and female samples - individuals that were found positive for the SRY gene were males as this particular gene is present only on the Y chromosome, making individuals that did not have an SRY gene females- and the phenotypes of the unknown donors were predicted. As far as the unknown samples were concerned by simply examining all the data available predictions of the donors' sex were made. Sample 4 and 5 was found to be males and sample 6, 7, 8 belonged to female individuals. There was a number of samples that based on the tests carried during the practical class their donor's sex was easily and correctly defined. Such samples were the ones numbered as 5, 6 and 8. (Question 1 and 2)
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However, some of the results available were to some extent different from the expected ones. For instance, sample 4 appears to belong to phenotypically female person although this particular individual had a genotype of a male. Also, sample 8 although having a male genotype, the donor's phenotype was of a female person. If it is assumed in this case, that no sex chromosome rearrangements took place in these individuals, then a possible explanation would be that something during the procedure of obtaining the results must have gone wrong. To be more specific, contamination could have altered the results of PCR, the process of amplification of the DNA samples might not have worked completely leading to results of low quality or complete suppression of the whole process might have resulted to uncertain data available for predicting the sex of particular individuals. (Question 3)
There are many known causes of sex reversal in individuals. Most of them involve chromosome translocations and generally mutations that occur on sex chromosomes. As it is known, due to the difference in length between the X and Y chromosomes in males, recombination and pairing is not possible to occur fully. However, the presence of two pseudoautosomal regions, PAR1 and PAR2, allows the two processes of meiosis mentioned earlier to be carried out successfully.
In cases, however, where chromosomes undergo recombination in the region outside the PAR1 gene, translocation of important sequences of the Y chromosome will take place and these sequences will then be located on the X chromosome. Similarly, X-specific regions will translocate onto the Y chromosome. G-banding is a method used by scientists that allows sex determination of individuals. However, due to the small size of the chromosome pieces that translocate, the results might not be able to show the true genotype of the individual tested. Individuals carrying this type of chromosomal rearrangements are defined by a sex reversal meaning that their genotype does not reflect their phenotype. XX individuals who are phenotypically males and XY individuals with a female phenotype are rare but both of them result in these reversed sexes due to chromosomal translocations of the SRY gene. Donor 8 must have undergone this kind of chromosomal rearrangement resulting to a female phenotype even though genotypically is a male (probably suffering from XY gonadal dysgenesis, also known as Swyers syndrome). This person possibly does not have the SRY gene or regions which are specific to the Y chromosome due to anomalous recombination events between the X and Y chromosomes. However, it should be taken in mind that even though PCR is a reliable process, errors might have occurred. (Question 4)
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(Please find the answer to the fifth question on a separate sheet attached.)
The translocation event must have occurred to the father involving his meiotic cells of his germline in order to result in individuals with X-Y translocation. (Question 6)
The PRKX/PRKY genes are known to consist of various open reading frames (ORFs) that code for a protein kinase. This kinase must have caused a mutation in this case, leading to the inhibition of the SRY gene as the ORF that codes for the protein kinase is located downstream of the SRY gene. This further leads to the formation of ovaries which explains the sex reversal in XY individuals. It should be also mentioned that the mutation affecting the SRY gene must have been a point mutation, such as insertion, deletion or base substitution (affecting the 2nd or 3rd base of the amino acid) affecting the fathers germline or caused in the very early development of the donor.
(The diagrams to the seventh question can be found on a separate sheet.) (Question 7)
The fates of the chromosomes that are produced after a recombination event will involve their breakage. More clearly, during anaphase II of meiosis the separation of the centromeres occurs which leads to the separation of the sister chromatids that belong to each chromosome resulting to their movement towards the poles of the spindle. Each sister chromatid will then exist as a separate chromosome. However, if it is assumed that an abnormal recombination event occurs, the chromosomes will undergo breakage. (Question 8)
Phenotypic males with a 46,XX chromosomal composition are mostly infertile. Formation of small testes characterizes also the phenotype of these individuals. Almost most of them have an SRY gene which is present only on one of their X chromosomes. Swyers syndrome individuals are also infertile; they appear to not have the SRY gene on their sex chromosomes. The infertility of the X translocation individuals can be explained by the fact that the gene responsible for the formation of testis is not fully expressed and as far as the infertility that characterizes the Y translocation individuals it is generated due to the missing SRY gene. Therefore, ovaries and testis in both cases cannot be fully made in the individuals that their sex chromosomes have undergone such chromosomal rearrangements. Lastly, small testes present in 46,XX males and streak gonads present in 46,XY females lead to a abnormal development of genitals in both cases that further result in the inability of the individual of being fertile. (Question 9)
Experiment 3- Genetics of a temperature sensitive mutation.
Stock A flies-both females and males- contained the gene that makes them temperature sensitive. When exposed to a specific temperature their eye shape seems to change dramatically resulting in bigger eyes than the wild type flies. Stock C female and male flies had a red slit-eye shape and they were not heat sensitive. The results from a cross that took place between stock A and C showed variable results. For instance, when A-females were crossed with C males the female in the offspring appeared to have a kidney shape-eye shape and they were not heat sensitive. The F1 males had big round eyes and they were temperature sensitive. In addition, when stock C females and A males were crossed, the female offspring had kidney eyes and showed no heat sensitivity and the male offspring had bar looking eyes and behaved in the same way as the females of the cross as far as temperature sensitivity is concerned. Therefore, the genes regarding the heat sensitivity must be sex linked and recessive firstly because the Stock A progeny was heat sensitive whereas the Stock C progeny was not and as a result only the females of the Stock AxC males cross appeared to have inherited the para(ts) mutation. As far as the eye shape genes are concerned, they were inherited in an autosomal semi-dominant mode. More clearly, there is a big heterogeneity between the phenotypes regarding the eyes of the two sexes of the offspring therefore the genes responsible in this case are inherited in an autosomal mode as not specific patterns of inheritance present in these particular crosses propose se-linked inheritance. The semi-dominance can be also explained by the heterogeneity in the phenotypes as three different types of eyes are present; kidney, bar and round eyes.
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(Please find attached on a separate sheet the diagrams) (Question 1)
As far as Stock B is concerned, the female and male progeny seem to contain a particular chromosome arrangement leading to an unusual pattern of inheritance regarding the mutation affecting specific phenotypes. Also, some of the flies contain the para(ts) allele and some of them not while at the same time the stock is true-breeding. When a stock is defined as true-breeding, it means that the phenotypes or more accurately each different allele present in the progeny will be inherited unaffected in the offspring. When flies from Stock B mated with files from Stock C this unusual pattern of inheritance was present in the offspring. More clearly, the phenotypes indicated that from a B-females x C-males cross all the F1 females inherited the phenotypes of their mothers while the F1 males inherited phenotypes from their fathers. Moreover, the F1 females and males from a C-female x B-male cross were found to present phenotypic characteristics from both of their parents with the mothers' phenotype to be present more in the offspring. The wild type is therefore more "persistent" in the offspring even though it is a true breeding stock and the phenotypes must have been inherited unchanged. (Question 2a)
Chromosome X and Y are the two distinct chromosomes that determine the sex in Drosophila melanogaster. Males are recognized by their XY constitution and females by their XX. The sex chromosomes contain most of the time genes that are not sex-specific. These kinds of genes have alleles mostly on the X chromosome and their mode of inheritance is therefore sex-linked. There are cases where XXY and XO flies exist. The first one is considered a female due to the number of X chromosomes (fertile) and the second a sterile male as the Y chromosome is important for fertility. In the case of a XXX female fly this particular constitution results in the death of the fly. The reason for this is because it is not possible for the X-chromosome to be inactivated during transcription. Also, the YO constitution is lethal because as mentioned before the X chromosome carries the most important genes. The stock in this case is considered to be a true-breeding one meaning that every single fly must be homozygous for an allele. Also, another kind of true-breeding could be true in this case such as a culture having abnormal chromosomal arrangements. The culture will then breed according to true-breeding technique but the flies used will not be homozygous but they will contain lethal genotypic arrangements. The stock therefore in this case is a true-breeding stock because the female flies have a XXY genotype.
The para locus inheritance of A x C cross does not seem to be similar to the way this mutated allele is inherited in the B x C cross. (Question 2b)
The reason that Drosophila melanogaster is used very often in experimental procedures by scientists is due to the fact that when mutations occur in a single gene the flies produce a huge variety in morphology. This is therefore the reason that flies with different eye color, wing shape and bristle and eye shapes exist. The mutations leading to this wide variety of phenotypes are used as genetic markers. Also, when scientists want to perform a true-breeding culture these mutations help to simplify the genetic populations produced. (Question 2c)
When flies containing the para(ts) allele are heated, then apart from the temperature sensitivity that characterizes them, changes in their bristles, eye shape and body color occur. This mutation mostly affects the eye shape as it is found that the para gene encodes a voltage-dependent sodium channel. The transcripts of this gene are located in the cortical regions of the central nervous system of the fly and also in the eyes. When the para locus is mutated therefore the channels that are encoded by it undergo functional and structural changes resulting in changes in the fly's phenotype. (Question 3a)
The protein that the para gene encodes has been found to be similar in structure and amino acid composition to the proteins of vertebrate sodium channels. These sodium channels are present in the neurons of Drosophila and different types of them result from alternative splicing of the para transcripts and further in many different phenotypes. (Question 3b)
The type of mutation that have occurred in order the para temperature sensitive allele to be produced must have been a point mutation such as base substitution, deletion or inversion on the gene as it still functions but it confers a different phenotype to the fly. (Question 3c)
The para gene encodes sodium channels as mentioned earlier which are affected by the changes in temperature and it is a reversible change as when it is affected paralysis of the neurons occurs which can further return to its initial stage. (Question 3d)
In the case of a fly homozygous for the null allele of the para gene its phenotype would be lethal or not affected at all meaning that it will presented by the wild type.