Robin L. Davies, Associate Professor of Biology

The Human Genome Project is a world-wide, collaborative effort to map and sequence all twenty-four human chromosomes (1-22, X and Y). This research is being pursued by individuals working in laboratories at many different kinds of institutions, including large research universities, medical schools, biotechnology companies, and non-profit research institutions. An examination of the list of institutions at which human genome research is being pursued would soon reveal that liberal arts colleges are largely missing from the list. However, at Sweet Briar, a number of women have been working on the Human Genome Project for the last two years. The following is a progress report on some of that work.

Specific Goals of the Project

The primary goal of this project is to construct a high-resolution physical map of a gene-rich region of human chromosome 12, namely 12p13. This physical map will take the form of a "cosmid contig", a contiguous set of overlapping recombinant DNA clones. Cosmids are artificial DNA vectors which can carry approximately 45,000 base pairs of the DNA of interest. This cosmid contig will be a valuable resource for the study of genome structure in the 12p13 region (see Figure 1). It will make possible the concerted sequencing of chromosome 12 DNA in this region, as well as providing reagents for the generation of new markers and for the localization of genes. We are concentrating on the 12p13 region because it is known to be a gene-rich region. I have experience working in the region, as I mapped the gene for a glutathione S-transferase enzyme to 12p13 while I was on sabbatical at the Albert Einstein College of Medicine (AECOM). An especially exciting aspect of concentrating on the 12p13 region is that some preliminary sequence information generated by my collaborator at AECOM, Dr. Kate Montgomery, suggested that a previously-unknown gamma-aminobutyric acid (GABA) transporter gene would be found in the 12p13 region. As GABA is an important inhibitory neurotransmitter in the brain, the discovery of a novel GABA transporter would be an extremely significant finding.

Dr. Montgomery constructed a physical map of 12p13 in large DNA vectors called yeast artificial chromosomes (YACs). YACs are valuable tools for physical mapping because of the immense amount of DNA they carry (from 100,000 to 2,000,000 bases). However, they are simply too large to be used for sequencing of DNA by current methods. The first goal of our project is thus to construct a higher resolution physical map using the smaller cosmid DNA vectors, because cosmids are excellent substrates for sequencing. An additional goal of our work is to link our cosmid-based map to the YAC based map, thus insuring integration of the sequence with the large-scale map of the entire chromosome.

Progress to Date

Because of the density of known markers in the 12p13 region, and the desirability of linking the cosmid-based map to the large-scale map, we decided to begin the selection of cosmids based on 16 known polymerase chain reaction (PCR) markers at the terminus of 12p13 (see Figure 1). Dr. Montgomery labeled the PCR primers for the known markers with radioactive phosphorus (32P) and used them as probes to identify cosmids from the chromosome 12-specific cosmid libraries. One hundred and ninety-six positive cosmids were thus identified. The positive cosmids were removed from the frozen library stocks and transported to Sweet Briar. Here we grew the cosmid-bearing bacteria on petri dishes and prepared frozen reference stocks prior to the isolation of cosmid DNA. To date our group at Sweet Briar has isolated DNA from 181 of the 196 positive cosmids. The next procedure we undertook was the determination of a restriction enzyme "DNA fingerprint" of the cosmid DNA, to determine that each cosmid was complete and to eliminate any duplicates. Thus far we have performed fingerprinting on 115 of the DNAs isolated, using the restriction enzyme Eco RI, and have detected only ten duplicates and six incomplete cosmids. Complete, unique cosmid DNAs were then subjected to PCR. So far we have performed PCR on 65 of the cosmid DNAs and have already begun to obtain results. Our first result from the PCR of the cosmid DNAs was the unambiguous placement of one marker, D121987, on the large-scale physical map (see Figure 1). This serves to demonstrate the advantages of working with the cosmid vector, which carries much smaller pieces of DNA than does the yeast artificial chromosome (YAC) vector which was used to make the large-scale map. Our next result was the construction of a preliminary cosmid map around the marker D12S994 (see Figure 2). These results have been communicated via electronic mail to our collaborators at AECOM. I expect the generation of the partial cosmid contig maps centered around each of our 16 markers to continue through 1997. At that point, the project will proceed in two steps.

1. Closing the gaps in the terminal region of 12p13.

Because of the number of cosmids we have already selected and the density of the markers in the terminal region of 12p13, it is likely that the individual marker-centered cosmid maps will overlap. Thus, we expect that completion of the 16 individual cosmid contig maps should result in the region being mostly or completely covered by one large contiguous cosmid map. The reasoning behind this expectation is as follows: The marker density in this region is approximately one marker per 60 kilobases of DNA. The size of the chromosome 12 DNA carried by each cosmid is on the order of 45 kilobases. If the leftmost and rightmost cosmids of the individual contigs overlap by less than 15 kilobases, each cosmid contig will cover 60 kilobases or greater, which should allow adjacent cosmid contig maps to overlap each other with no gaps. If gaps do remain between any adjacent cosmid contig maps, we expect that they will be small and we will proceed to close the gaps. This will be accomplished by taking the ends of the cosmids flanking the gaps and then using them to go back to the original cosmid library to select new cosmid clones, in the expectation that at least half of them will extend into the gap. These new cosmids will be placed in relation to the existing cosmids in the map, and the procedure will continue until all of the gaps are closed, at which point approximately one-sixth of the entire 12p13 region will be mapped. The results of this work will be forwarded to our colleagues at AECOM, who will use the information to guide the sequencing effort. Our work will be published as part of journal articles prepared in collaboration with the chromosome 12 team at the AECOM/Yale Genome Science and Technology Center.

2. Mapping additional sections of 12p13.

When all of the gaps in the terminal region are closed, or if no gaps are found when all 16 marker-based cosmid contig maps are complete, we will continue our coverage of chromosome 12p13. We predict an increased rate of progress after this first region has been completed because we will be working with established protocols adapted to our particular laboratory environment. It is reasonable to expect that we will be able to cover one-half of 12p13 which would represent approximately 5% of the entire chromosome. This will be a significant accomplishment for two reasons: 1) the cosmid map will serve as the substrate for the automated sequencing of this portion of human chromosome 12; and 2) this region is expected to contain at least 200 genes, only a few of which have been mapped, and the availability of the cosmid clones will speed gene discovery.

Personnel

Several Sweet Briar students have contributed to the Human Genome Project research to date: Ann Kays '96, Cassandra Thomas '97, Nicole Kelleher '97, Susan Bobb '00, Amelia Scott '00, Stephanie Belk '98, Katie Wood '01. This summer, the team included Natalie Lindfors '98, Julissa Yabar '00, Katie Wood '01, and Tamara Trout '01.

Figure 1. A section of human chromosome 12p13. The sixteen PCR-based markers used in the current study are shown above the horizontal line representing a portion of chromosome 12p13. Five cosmids already placed on the map are shown below the horizontal line. The cartoon of chromosome 12 below the cosmids indicates the region under study.

Figure 2. A partial cosmid map centered on the marker D12S994. The horizontal lines represent cosmid DNAs, with the names of the cosmids appearing to the left. The solid vertical lines represent Eco RI cut sites, and the sizes of the fragments which result are given in basepairs at the top of the figure. The dashed vertical lines represent Bam HI cut sites, and the sizes of the fragments which result are given in basepairs at the bottom of the figure.