Radio-labeled YAC DNA was initially hybridized to high density gridded filters from a flow sorted chromosome 1 cosmid library, after suppression of repetitive sequences (see materials and methods, section 2.2.9). Although the ultimate strategy was based on constructing the majority of the map in PI artificial chromosomes (PAC), using YAC to PAC library hybridizations, the cosmid data provided an additional lq21 resource.
Each YAC was hybridized to cosmid filters individually, table 3.1 indicates the counts per minute per ml of labeled YAC (as determined by a Beckman scintillation counter) used in each hybridization. Cosmids were scored and the data was entered into the xv2well program (Unix operating system) written by Huw Griffiths, ICRF, London (unpublished). Cosmid addresses for each YAC hybridization were calculated and compared using a computer program written by Colin James in Dr Nizetic’s laboratory. The program generated a table displaying each positively scored cosmid with respect to the YAC used in each hybridization. Cosmids positive with more than three YAC clones, or two or more overlapping YAC clones (i.e. 764_a_l and 950_e_2), were scored as non-specific.
From this data each positive cosmid could be assigned to a ‘bin’ or ‘pocket’, depending on its’ hybridization pattern. A total of 13 pockets were produced containing 588
specific cosmids. Figure 3.2 displays these pockets in relation to the YAC clones. Two anomalies are seen when these data are compared to the Marenholz YAC contig: YAC clones 907_e_6 and 874_d_5 hybridize to a number of cosmid clones uniquely, where the Marenholz data displays these clones overlapping. One explanation for this would be the fact that the YAC clone 874_d_4 is known to be re-arranged (Marenholz et al,
1996), while the YAC clone 907_e_6 could possibly contain chromosome 1 specific repeats (Zhang et al, 1999) generating a large number of uniquely positive cosmid clones within the library.
Independent screening of the cosmid library by two laboratories studying individual genes within the EDC had identified a number of cosmid clones. These cosmid clones were present within the pockets as indicated by figure 3.2.
CEN
M—
Okb 50p kb IQOOkb I500kb lOOOkb 2 3 0 0 k b 3000k b TEL
-► .s'* SW > \r so oc 25 17 o I ---1 I 764_a_l I . 1 ____________________1 30 27 I 907_e_6 I J __________ I CD 200 ★ +43 r ---, I 874_d_5 I 1_______I 22 O
o
I 9 5 5 _ e_ ll I 94 22 I 950_e_2 I . I--- J 82Figure 3.2. Cosmid pocket map generated using 5 YAC clones covering approximately 3 mega-bases of human chromsome lq21. The top horizontal bar displays the scale in kilobases and the orientation (CEN = centromere, TEL = telomere). The thicker horizontal bar below the scale displays a schematic representation of the chromosome with the position of genes and markers indicated by circular shapes (THH - trichohyalin, FLG - pro-filaggrin, INV - involucrin, LOR -loricrin). White circles depict genes used to screen the flow sorted chromosome 1 library by independent laboratories (Dr J. Ragoussis and Dr. D. Hohl). Below the chromosomal bar are horizontal lines representing YAC clones. Dashed lines depict the relative positions of YAC clones as determined by Marenholz et al, 1996 (not to scale) while solid lines depict the relative positions of YAC clones used to generate the pocket map (not to scale). Vertical lines display the relative positions of the cosmid pockets (determined by the hybridisation pattern of each individual YAC clone). The number of cosmids is indicated for each pocket. In the case of YAC 874_d_5, ★ indicates the number of cosmids uniquely positive for this clone (43). Circular shapes present on YAC clones or within pockets indicate that either the YAC clone is positive for the corresponding marker/gene PCR (grey circular shapes) or cosmids that have been previously identified with hybridisation screens of the library with the particular gene (indicated by white circular shapes) are present within the individual pocket.
Interestingly the pocket data revealed that the YAC clones 907_e_6 and 955_e_l 1 overlapped. This had not been indicated in the map produced by Marenholz and co workers, presumably due to the lack of markers for this interval. This enabled the number of YAC to PAC hybridizations to be reduced from five to four by excluding the now redundant, rearranged YAC 874_d_5.
Labeled YAC DNA was hybridized to high density gridded membranes (or filters, herein) of the PAC library RPCI-1, representing the whole human genome. In addition to suppressing human repetitive sequences within the labeled DNA (prior to YAC to PAC hybridization as described), around 1 pg of PAC vector DNA that contained no cloned insert was also added to the competitive hybridization reaction. This was due to the fact that about one quarter of the clones within the library RPCI-1 contain no insert (HGMP Resource Centre, Hinxton, UK), and that a number of these PAC clones have been scored positive with other YAC to PAC hybridizations (Jurgen Groet, Dr Nizetic laboratory, personal communication). Even after this, a number of positive PAC clones were seen that did hybridize with other YACs from unrelated regions of the human genome (J. Groet, personal communication). After removing these non-specific clones, a total of 105 were scored. These PAC clones could be ordered into seven pockets determined by the individual YAC hybridization patterns.
Figure 3.3 displays the numbers of positive PAC clones positioned within these
described pockets. A number of differences can be seen between the cosmid pocket and the PAC pocket maps after excluding YAC 874_d_5 from the cosmid pocket data. The factor between literal numbers of clones for each pocket shows a wide variance. In the case of YAC 764 _ a_ l, 34 uniquely positive cosmid clones are seen in comparison to 21 uniquely positive PAC clones, which produces a factor of 1.62 between the two figures. In the case of the pocket produced by YAC clones 955_e_l 1 and 950_e_2, 22 cosmid clones and only 2 PAC clones are present; a difference by a factor of 11. In order to assess the accuracy of the PAC pocket data before proceeding with constructing the bacterial map, the expected number of positive PAC clones for each YAC clone in question was determined.
CEN Okb M— L- 500kb lOOOkb 1500kb 2000kb TEL I ^ o 764 a 1 955 e 11 907 e 6 950 e 2
Figure 3.3. PAC pocket m ap generated using four Y AC clones covering approxim ately 3 m ega bases of hum an chromosomal region lq21 (of which only 2 megabses are shown). The top horizontal line displays the scale in kilobases and orientation (CEN = centromere, TEL = telomere). The horizontal bar below this shows a schematic representation of the chromosome displaying the position of three genes (rhomboid shapes) used to verify the Y AC content by PCR (IVL = involucrin). Horizontal lines below this depict the Y AC clones indicated (not to scale). Rhomboid shapes present within these YAC clones indicate the presence of the respective gene within the Y AC clone as determined by PCR. Vertical dashed lines delineate the PAC pockets which are represented by black or white rectangles at the bottom of the figure. Numbers present in these rectangles indicate the number of PAC clones scored positive within the particular pocket.
The average length of a PAC clone from the RPCI-1 library seen when working on human chromosome 21 (Groet et al, 1998; personal communication) is 1 lOkb. The RPCI-1 library was constructed to yield a five fold coverage of the human genome (loannou et al, 1994) but the revelation that only 75% of the clones contain a cloned insert (HGMP Resource Centre, Hinxton, UK) reduces this to 3.75 fold. W ith these statistics in mind, and assuming the library coverage is equal genome wide, the expected number of positive PAC clones from a labeled YAC o f known size can be worked out using the formula:
YAC size in kb x 3.75 (library coverage) = expected number of positive PAC clones 110 (average PAC size in kb)
Table 3.2 displays the predicted number of PAC clones (as determined with the above formula) and the actual number of PAC clones scored positive with the labeled, PFGE sized, YAC clones.
Table 3.2. Predicted compared to actual number of positively scored, specific, PAC clones identified with four YAC clones (indicated) used as hybridization probes against the total human PAC library, RPCI.
YAC SIZE in kb PREDICTED ACTUAL
764_a_i 900 30.7 28
907_e_6 1040 35.5 35
955_e_ll 1125 38.4 32
950_e02 680 23.2 22
As the degree of overlap between YAC clones is not known the expected distance covered by the positive PAC clones can be worked out from the above formula using the known value for the positive PAC clones (105).
Estimated distance covered in kb = number of positive PAC clones x 110 3.75
The number of PAC clones identified from the RPCI-1 PAC library was in good agreement with the predicted number of PAC clones (table 3.2). Therefore, the 105 positively identified PAC clones were selected to constitute the ‘lq21 sub-library’ - a library of recombinant bacterial clones enriched for human chromosomal region lq21, specifically the Epidermal Differentiation Complex.