LA TROBE UNIVERSITY Bachelor of Biomedical Sciences (GEN2HG!
"roso#hila Re#or$
Done by: Grace Chan Wai Man (18884077) Lab partner: Heng Jun Hao (1117!71) Lecturer: Dr "#e$i% Chon Date %ub&itte' :!811!01
The allele that is most likely to be dominant is the w+ (wild type) allele. The recessive allele is the w (null mutant) which were located at the X chromosome. Therefore two copies of the w allele (null mutant) was required in order for the gene to be expressed in female flies. ale flies which are hemi!ygous only require one w allele to express the traits of the recessive allele. "or example# when a fly had a genotype of $bw+ bw+% st st% w &'# the eye color that is being expressed in this fly is white color due to the w gene present in the X chromosome. This is an example of epistasis# of which the expression of a gene is affected by the presence of other genes. ("igure )
Figure 1 show the genotype of flies which express the phenotype for white eyes. ale flies
which are hemi!ygous require only one mutant X chromosome to express this recessive gene.
Cross 1: red-eyed females and white-eyed males
*hite eyes due to scarlet and brown mutations + + bw % st
bw% st +
+
+
+
bw % st
+
bw bw
bw bw
white
&
w +
w w
+
w
+
w w
+
w
w
+
&
+
st st
,
bw% st
*hite eyes due to mutation
st st
+
+
,
+
bw bw
bw bw
w
+
&
+
st st
st st
-xpected percentages of each type of
-xpected percentages of each type of progeny
progeny
,
/0
,
/0
/0
,
/0
/0
,
/0
/0
/0
Cross 2: white-eyed females and red-eyed males
*hite eyes due to scarlet and brown mutations bw% st
+
+
bw % st
bw bw
bw bw
bw % st +
+
st st
,
bw% st
+
bw bw +
st st
+
+
*hite eyes due to mutation
+
w
white +
w +
w w
& w &
+
st st +
bw bw +
st st
,
w
+
w w
w &
-xpected percentages of each type of
-xpected percentages of each type of
progeny
progeny
,
/0
,
/0
/0
/0
,
/0
,
/0
/0
/0
The real outcome obtained from the cross 1 and cross 2
The actual outcome of cross ("igure 1 )
,
/0
,
/0
/0
/0
The actual outcome of cross 1 ("igure 2 )
,
34.0
,
/0
/0
2.50
6ross is the cross between the redeyed females and whiteeyed males. The expected ratio for this cross is that /0 of the flies will be redeyed males and the other /0 will be redeyed females. 7n the actual cross# the percentage of the redeyed female flies and the percentage of the redeyed male flies were each found to be /0 of the total number of flies. Therefore the data achieved is the same as the predicted values in both the autosomal and sex chromosomes. *hen a reciprocal cross was carried out# the percentage of getting redeyed male and female flies in the inheritance color gene on the autosomal chromosome were /0 each. *hen the w and *+
alleles of the eye color gene were taken into account# we can get the predicted data as //0 of the female flies will have red eyes and //0 of the male flies will have white eyes. "rom the actual result# //0 of the female flies had red eyes and //0 of the male flies had white eyes. This confirmed that the gene for eye color in the flies that had an epistasis effect on both the red and brown eyes of the flies was located on the x chromosome of the flies. "rom the data# since all the male progeny of the " generation for the 1 nd cross had white eyes therefore the gene encoder for the membranebound transporter protein was located at the X chromosome and is an Xlinked recessive gene.
PCR analyses The 89: of the parental flies and the progeny flies are taken and undergo ultiplex ;6< reaction that amplified more than one product by introducing different types of primers. 7n this experiment two type of forward primers were introduced. =ne was the wild type primers and the other one was the mutant type primers. ?bp and the second one was the mutant type white locus gene. 7ts ;6< products length were predicted to be approximately 5?35bp. 6ompared with the blast result# the wild type gene length was almost similar to the wild type gene length in the ;6< which was almost >//bp. ("igure 3 and "igure ) This is because no
drastic mutation occurred in the wild type gene of the flies. *hile comparing the gene length of the mutant white gene# the expected ;6< length was 5?35bp# but the actual gene length for the white gene was only //bp. Therefore there are some mutations occurred in the white gene locus. "or example deletion mutation may have occurred in the white gene and reduced the length of the actual white gene that were obtained during the ;6< process. ("igure 3 and "igure 4)
*igure + %ho,% the re%u#t o- the .ri&er/#a%t on#ine -or the W pri&er an' pri&er ,hich %ho, that the #ength o- the .C pro'uct% ,a% e$pecte' to be 87bp2
*igure %ho,% the re%u#t o- the .ri&er/#a%t on#ine -or M pri&er an' pri&er ,hich %ho, that the #ength o- the .C pro'uct% ,a% e$pecte' to be 8748bp2
Sequence analyses
The @last program was used to compare the sequence of the wild types and mutant products. "igure ? and 5 showed the result of the blast by inserting the wild type ;6< products sequence into the program. The result of the blast showed that there are //0 similarities between the wild type ;6< products and the wild types gene from the database. The query sequence to 51 matches the sequences of the ;6< wild type products from nucleotide sequence to >311 completely. Therefore# no mutation occurred on the white locus gene. ("igure ? and 5) The sequence for the mutant ;6< products was then inserted into the blast program and the result online were screenshot and labelled as figure > and / below. "rom the program the query sequence from to 2/3 matches with the mutant ;6< products. Atarting from query 2/ to 21# deletion and insertion mutations occurred within this alignment sequence. "rom the query sequences 214 onwards to 323# the sequence of the mutants ;6< products sequences matches the database sequence from 32 to 212 except on query sequence 223. =n query 223# substitution mutation occurred and change the original Buanine to Thymine. The deletion# insertion and substitution mutations caused the loss of gene function and cannot produce the membrane transporter protein for receiving the pigments synthesis from the scarlet gene and brown gene. ("igure > and /)
*igure 7 an' 8 %ho, the co&pari%on o- ,hite gene ,ith the ,i#' type .C pro'uct%
*igure an' 10 %ho, the co&pari%on o- ,hite gene ,ith the &utant type .C pro'uct%
Conclusion
The eye color of the drosophila melanogaster was controlled by 2 types of genes located in both the autosomal and sex chromosomes. *hiteeye flies will express this phenotypes if there are both mutated w gene in the X chromosomes. "or example whiteeyed female flies contain 1 alleles of w mutated gene in both their X chromosomes. @ut for male flies the due to being hemi!ygous# one allele of w mutated gene was required to express the white eye trait in the male flies. @ut when there were no mutations occurred on the X chromosome white gene locus# the flies will still express the whiteeyed phenotype if both the autosomal genes on chromosomes 1 and 2 were being mutated. This is because the gene making both the xanthommatin and drosopterin were mutated and thus the eyes of the flies appeared to be white. The deletion mutation in the white gene will lead to loss of function of the gene and thus the wild white gene that are responsible for encoding the transporter protein in membrane cannot synthesise the protein detecting the pigments synthesis by the gene located at chromosomes 1 and 2.
!tended questions
) "lies that are homo!ygous for a small deletion in the promoter of white that impairs <9: polymerase binding. *hen mutations occurred on the promoter site it may alter the expression of the gene and hence has a lower transcription of the membrane transporter protein. Therefore the numbers of membranes transporter protein decreases the detection of the pigments from the transporter. Therefore the eye color of the flies will only express /0 of the normal amount of the pigment produced by the normal scarlet gene or the normal brown gene of the flies. 1) Cetero!ygous flies with missense mutation of the coding sequence of scarlet gene will have normal gene encoding for brown pigment and the mutated gene that will produce nonfunctional protein. The ubiquitination and proteosomal degradation will degrade the nonfunctional protein. 8ominant alleles which is the wild type scarlet gene will thus produce the brown pigment for the eye of the fly. Aince wild scarlet gene is dominant in producing brown pigment# the flies that had this mutation will then had brown color eyes.
A##endi%
Figure 2: " flies from cross # between homo!ygous redeyed females and homo!ygous white
eyed males. 6ircled are the female flies.
Figure ": " flies from cross 1# between homo!ygous whiteeyed females and homo!ygous red
eyed males. 6ircled are the female flies.
*igure 4 %ho, the actua# .C re%u#t -or the *#ie% D3"
5ub%tituti
0+/!+ 'e#etion an' in%ertion
Figure #: 6hromatograms showing part of the data from sequencing wild type and mutant ;6<
products
