Spectral Karyotyping (sky) Establishes Chromosomal Homologies between a New World Monkey (pithecia Pithecia) and Humans (homo Sapiens)

Inspired by the already established chromosome homology between certain New World Monkeys and humans (Stanyon, et al., 2004), this cytogenetic study was designed to produce a homology map comparing the chromosomal arrangements of the Pithecia pithecia (White-faced Saki) and human karyotypes. After metaphase harvest and G-banding, a P. pithecia karyotype was constructed. Fluorescence in situ hybridization was then performed on a set of P. pithecia metaphase chromosomes using a Human Spectral Karyotype Reagant kit. Spectral hybridization allowed for the production of a P. pithecia karyotype labeled with a mosaic of colors representing the twenty-four human chromosomes. The extent of homology found is striking. Ten probes hybridized specifically with single sites on P. pithecia chromosomes, while the other fourteen human probes hybridized either with multiple P. pithecia chromosomes or in tandem arrangements along a single primate chromosome. The homology map illustrates the significant chromosomal conservation that links these two evolutionarily distant primates. INTRODUCTION The disruption of linkage groups by chromosomal rearrangements can present barriers to the interbreeding of two populations and facilitate subsequent speciation. These chromosomal changes can be demonstrated through cross-species painting (Ferguson-Smith et al., 2005). Homologies have been established between humans and many Old and New World monkeys and even some prosimians. Primate species that diverged approximately fifty million years ago show homologies of entire chromosomes. The present investigation focuses on the Pithecia pithecia (White-faced Saki), a New World Monkey (infraorder Platyrrhini) of the family Cebidae and subfamily Pitheciinae. New World Monkeys are estimated to have diverged from the ancestral primate almost forty million years ago, and about thirty-five million years before the divergence of hominids. Cross-species painting is most often accomplished by single chromosome hybridizations. However, some studies using spectral karyotyping with multiple probes produce results comparable to the more labor-intensive single chromosome paints (Best et al., 1998; Rens et al., 2001). The ASI Spectral View Imaging System produces human karyotypes in which each chromosome is painted a distinctive color. The process of spectral karyotyping (SKY) involves the hybridization of a set of whole chromosome painting probes, each labeled with a distinctive subset of one to four dyes from a set of five fluorochromes. This creates twenty-four distinct fluorochromosomes that serve as probes. Hybridization of each fluorochromosome is determined by the spectra detected at each pixel in an image field using an interferometer (Schrock et al., 1996) Spectral karyotypes of metaphase chromosomes from primates cross-painted with the ASI Human Spectral Karyotype Reagent Kit provide maps of homologies to human chromosomes in a single hybridization. A homology map, or color-coded diagram showing the chromosomal organization of the primate karyotype with respect to human chromosomes, is the major illustrative method of reporting cross-species homologies. Such homology maps have been published for representative species of the other Pitheciinae genera, but not Pithecia (Stanyon et al., 2004). C. MULLIN: SPECTRAL KARYOTYPING -2After successful spectral hybridization, the resulting homology map showed extensive chromosomal conservation between the Pithecia pithecia and human karyotype. Both conserved and derived chromosomes were traced back to their initial states in the karyotypes of common ancestors from the major primate groupings, in order to contrast the evolution of the P. pithecia and human karyotypes. The mapping results were then compared to a similar cross-species study by Stanyon, et al. (2004) in order to illustrate the common chromosomal links between the Chiropotes and Pithecia genera of Pitheciinae. MATERIALS AND METHODS Acquisition of cell line A Pithecia pithecia fibroblast culture PR00239 was obtained from the Intergrated Primate Biomaterials and Informatics Resource (IPBIR). Tissue culture The culture was transformed, maintained, and subcultured by the fibroblast tissue culture laboratory staff at Coriell Institute for Medical Research. Metaphase cell harvest When the cells appeared to be mitotic (round) under the inverted microscope, the culture was prepared for metaphase chromosome analysis by standard techniques. First, the culture was treated with colcemid to arrest cells in metaphase by disrupting spindle fiber assembly (mitotic arresting). The resulting monolayer of cells was detached from the flask by brief treatment with warm (37o C) Puck’s Versenetrypsin (0.02% EDTA, 0.041% trypsin) and this suspension was centrifuged for 8 minutes, supernatant poured off, and the remaining pellet resuspended in the small amount of residual fluid. 10 ml of hypotonic solution (2:1 sodium chloride:sodium citrate) was added to increase cell volume and allow for expansion of the mitotic chromosomes, and this suspension incubated at 37o C for 15 minutes. Next, prefixation was achieved by adding a pipette of cold fixative (3:1 methanol:acetic acid) to the tube, mixing gently by inversion, and centrifuging for 8 minutes. The resulting pellet was resuspended in the residual fluid, and 10 ml of the cold fixative added slowly with continued agitation. Fixation was allowed to proceed at 4o C for 15 minutes. After the first fixation, the tube was centrifuged for 8 minutes, supernatant poured off, and the pellet resuspended in 10 ml of the cold fixative. This fixation process was repeated two more times to ensure that the metaphase chromosomes were completely preserved, membranes and chromatin hardened, and all cytoplasm removed by dehydration. Fixed pellets could be stored at 4o C or used immediately for chromosome analysis. Slide preparation Slides were made in a Thermotron set to approximately 43% humidity to prevent overspreading. A wet, sterile slide was held at a 45o angle and 3 drops of the fixed pellet dropped across the upper edge of the transparent region, allowing the cell dilution to spread uniformly down the slide. Once dry, slides were examined under a phase-contrast microscope setting to check for chromosome spreading and mitotic index (abundance of metaphase cells) to insure viability for staining. G-banding To prepare metaphase chromosomes for staining, the slide was first aged on a slide warmer at 55o C overnight, then heated in the oven at 90o C for 1 hour before G-banding. After the one-hour cooling period, standard G-banding procedures involving trypsin treatement followed by Wright’s staining was performed. Following air-drying, slides were examined under the microscope to check for sharpness of banding. Karyotyping Using the microscope and computer imaging system, metaphase cell images with the best chromosome spreading, fewest overlaps, and sharpest banding were captured and analyzed. Homologous chromosome pairs from the metaphase image were arranged into a karyotype (by cutting and pasting) based on size, centromere position, and banding patterns. TCNJ JOURNAL OF STUDENT SCHOLARSHIP VOLUME X APRIL, 2008 -3Spectral Karyotyping (Fluorescense in situ hybridization) In order to denature outer chromosomal proteins and expose DNA, the slide was treated with pepsin, covered with a glass coverslip, and observed under phase contrast microscopy to note any remaining cytoplasm, debris, etc. If sufficient, the slide was then washed, incubated in 1% formaldehyde, and dehydrated in 70%, 80%, then 100% ethanol at room temperature for 2 minutes each. In order to denature the chromosomes before probe application, the slide was air-dried and then incubated in denaturation solution (35 ml formamide, 10 ml distilled H2O, 5 ml 20x SSC, pH 7) at 70o C for about 1 minute. The slide was quickly removed dehydrated and air-dried. Simultaneously, 10 ul of the Human Spectral Karyotyping probe was denatured by incubation in a water bath at 80o C for 7 minutes. It was imperative to protect the probe from direct light and store at 20o C until ready for use. Once the slide was dry, the denatured probe was applied within score marks on the slide, covered with a precleaned coverslip, and protected by sealing the edges with rubber cement. The slide was then transferred to a hybridization chamber and incubated at 37o C for 36 hours. Under these conditions, denatured (single-stranded) human DNA of the fluorescently-labeled probe could interact and anneal with complementary regions of the denatured primate chromosomal DNA fixed to the slide. These hybridizations could then be observed and interpreted through the ASI Spectral Imaging System. Spectral imaging Each of the human chromosome probes of the Spectral Karyotyping Reagent was combinatorially labeled with a distinctive subset of one to four dyes from a set of five fluorochromes. Hybridization of each fluorochromosome was determined by the spectra detected at each pixel in an image field using an inferometer (Shrock, et al., 1996) in the ASI Spectral View Imaging System. This system allowed for the viewing and capture of metaphase chromosome hybridization images that were then arranged into a color-coded P. pithecia karyotype, or homology map, showing the organization of P. pithecia chromosomes with respect to human chromosomes. C. MULLIN: SPECTRAL KARYOTYPING -4RESULTS Karyotype of Pithecia pithecia A G-band karyotype of PR00239 was prepared (Figure 1) and compared to the karyotype for P. pithecia reported by Henderson et al. (1977), confirming that PR00239 was in fact a male P. pithecia (48, XY). Figure 1. A male Pithecia pithecia G-band karyotype prepared by trypsinization and banding with Wright’s stain. The diploid number (2n) is 48, with nine metacentric autosomal pairs and fourteen acrocentric autosomal pairs. The X is metacentric and similar in size and band pattern to most of the higher primates. The Y is acrocentric, quite small and non-descript, also comparable to other primates. TCNJ JOURNAL OF STUDENT SCHOLARSHIP VOLUME X APRIL, 2008 -5Hybridization of human probes to Pithecia pithecia chromosomes In Figure 2, the image of a full set of P. pithecia metaphase chromosomes hybridized with the twenty-four human chromosome probes of the Human Spectral Karyotyping Reagent Kit shows the spectral colors displayed. Figure 2. Hybridized P. pithecia metaphase chromosomes in spectral colors. The primary value of this image is to estimate the quality of hybridization achieved. Although spectrally distinct, some of the dyes and dye combinations are not discernable microscopically. To enable viewing, each pixel in the image field is assigned a classified color based on the spectra detected. Figure 3 shows the combinatorial table used by the interferometer to convert spectral to classified colors. C. MULLIN: SPECTRAL KARYOTYPING -6Figure 3. Combinatorial table used to determine hybridization of each fluorochromosome and convert spectral to classified colors. The spectral karyotype (homology map) produced after conversion to classified colors is displayed in Figure 4. The black and white reverse-DAPI images, which are comparable to G-banded chromosomes, allow for arrangement into a karyotype. Figure 4. Homology map showing the hybridization pattern of human chromosome probes within the Pithecia pithecia karyotype. Each color corresponds to a single human chromosome whose number is listed next to each color-coded segment. Black and white images show P. pithecia chromosomes in reverse-DAPI banding. 6