11. Anexos
11.10. ANEXO N°10 Categorización de la pregunta número 4
from Repeated Sub-Culture (300 Generations)
of A singe Parental Strains of
C. Albicans
A single colony of strain RIHO 30 taken from a YPD overnight-grown plate culture was inoculated into a tube containing 2 ml of sterile YPD medium. The culture
was grown at 37∘C for 12 hours. The cultures (10 𝜇l) were then inoculated into
another tube containing 2 ml of sterile YPD medium and the tube was incubated
for 12 hours or 24 hours. Ten 𝜇l of the cultures were then transferred again into a
new tube containing 2 ml of sterile YPD medium. The process was repeated until the cells reached 300 generations. After the incubation time, the optical density of the culture was measured using spcectrophotometer (Nova Tech.) at 600 nm to determine the number of cell generations. For the first 8 transfers, after the incubation time the optical density of the culture were measured using spcectrophotometer (Nova
Tech.) at 600 nm to determine the number of cell generations. The data were then used to calculate the number of transfers required for reaching 300 generations. Two
hundreds 𝜇l of the last culture were spread on a the surface of YPD agar plate, and
the plates were incubated at 37∘C overnight. The cells grown on the agar surface
were then transferred into a tube containing 5 ml of distilled water. The culture was diluted and spread on three YPD agar plates with approximately 100 cells per agar.
The plates were incubated at 37 ∘C overnight and 60 single colonies were randomly
chosen to determine allele size of the repeat regions ofYWP1,HWP1 and EAP1 by
genotyping. The 300 generation samples were prepared by Zhuo Zhou [52].
2.9
Statistical Analysis
Statistical analysis used in this study include the Chi-square tests and the t test. There are 2 Chi-square tests used: the Chi-square goodness of fit test, and the Chi-square test for contingency tables. The Chi-square goodness of fit test was used to check whether particular allele combinations or an alleles predominated in a group of strains compared to other allele combinations or alleles in the same group. Thus the test checks whether the frequency of a certain allele combinations or alleles is significantly higher than the observed frequencies of the other allele combinations or alleles. The Chi-square test for contingency tables was used to check whether the predominant allele combinations or alleles in a group of strains overrepresents compared to those particular allele combinations or alleles in the other group of strains. Thus it checks whether the frequency of predominant allele combinations or alleles in a group of strains is significantly higher than those particular allele combinations or alleles in the other group of strains. The t-test was used to check the significant difference between two means of the differences in the number of repeat units in the two alleles in an individual strain. The procedure of statistical analysis in this study follows the standard statistics textbook written by Sheskin [71].
Chapter 3
Alleles of the
YWP1
Gene
This chapter describes the results of the allelic characterization of the YWP1 gene
in strains of interest, which is aimed to determine whether YWP1 is a contingency
gene, i.e. whether YWP1 has a role in adaptation by changing the number of repeat
units within the coding sequences. For this purpose, the allelic distribution ofYWP1
in GPG and non-GPG strains, in commensal and infection strains, and in strains isolated from different sites of the humans body were compared one to another. For a contingency gene, GPG and non-GPG strains, two groups of strains with different genetic backgrounds, should select the same alleles, which are advantageous alleles. A
comparison of the allelic distributions of theYWP1 gene in commensal and infection
strains, and in strains isolated from different sites of the humans body, respectively,
is to observe whetherYWP1 acts as a contingency gene, when the C. albicans state
changes from commensal to pathogenic, and when it moves to particular sites of the humans body.
The chapter begins with a description of the identification of the repeat units in
YWP1, where two regions in the gene were identified to contain repeat units. This is
followed by two sections containing the results of the characterization of the YWP1
alleles for each repeat region. In the first section, since there was no variability in the number of repeat units observed, the analysis was not continued. The second
section contains the results of the allelic characterization of YWP1 for GPG and
non-GPG strains of infection strains, two groups of strains with different genetic
backgrounds, and the results of the allelic characterization for the commensal strains, for comparison with the infection strains. Analysis of the diversity of the alleles and allele combinations, and analysis of combination of the two alleles in an individual strain for all strains of interest are explained before the description of the results of
the allelic characterization of YWP1 for strains isolated from different sites of the
humans body. The chapter ends with a discussion of the biological implications of the results.
3.1
Identification of the Repeat Units in the
YWP1
Gene
The repeat units inYWP1 were identified using MacVector software (MacVector Inc,
www.macvector.com) to analyse the DNA sequence of YWP1 from strain SC5314,
downloaded from www.candidagenome.org. In MacVector, the nucleotide chain of a particular gene was arranged from the top to the bottom of the vertical axis, and from the left to the right of the horizontal axis starting from the 5’ end. This forms
a square matrix, and each edge repeats the length of theYWP1 sequence. With this
method, there are dots forming a diagonal line from top left to the bottom right. The line is formed by dots which are a consequence of the fact that at those positions, the vertical and horizontal axis refers to the same nucleotide. The presence of a symmetric pattern of dots in the matrix distinct from the diagonal line indicates
regions containing repeat units. The result of the DNA sequence analysis of YWP1
from strain SC5314 is shown in Figure 3.1.
Primers YWP1AF and YWP1AR (see Table 2.5 on page 24) were designed to amplify the region from nucleotide 607 - 1424 (818 bp), which is a region contain- ing repeat units, and for convenience is called repeat region 1. Primers YWP1BF and YWP1BR (see Table 2.5 on page 24) were designed to amplify the region from nucleotide 493 - 766 (274 bp), which is a region containing repeat units, and for convenience is called repeat region 2.
CHAPTER 3. ALLELES OF THE YWP1 GENE 31
Figure 3.1: Identification of the repeat unit in YWP1 using MacVector software
(top figure). The identification was performed on the DNA sequence ofYWP1 from
strain SC5314 downloaded from www.candidagenome.org. The grey and black thick lines indicate regions amplified, i.e. nucleotide 607 - 1424 (818 bp) for region 1 and nucleotide 493 - 766 (274 bp) for region 2 (bottom figure).