ABSTRACT
Chromosome-specific cosmid libraries are an extremely useful resource for positional cloning projects. Once a particular region of interest has been identified, it would be of value to have an approach for isolating chromosome band-specific cosmids that could be assembled into a sublibrary for rapid screening. We constructed a region-specific sublibrary of 700 cosmids by screening a chromosome 12-specific cosmid library with a complex probe generated by degenerate oligonucleotide-primed PCR of a microdissected homogeneously staining region containing sequences amplified from chromosome 12q13-q15. Based on fluorescence in situ hybridization, approximately 60% of the cosmids in the sublibrary were derived from the microdissected region. To demonstrate further the utility of this sublibrary, a 150-kb contig containing the SAS and CDK4 genes was constructed, as well as several additional contigs between CDK4 and MDM2. This study demonstrates the possibility of utilizing probes generated by microdissection for assembling band-specific sublibraries that are amenable to rapid screening with multiple markers.
Subject(s)
Chromosomes, Human, Pair 12 , Neoplasms/genetics , Proto-Oncogene Proteins , Base Sequence , Chromosome Mapping , Cosmids , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinases/genetics , DNA Primers , Gene Amplification , Genomic Library , Humans , Molecular Sequence DataABSTRACT
We have constructed and characterized two related human chromosome 12-specific cosmid libraries. DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector. Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LL12NC01 and LL12NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats. One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH). Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries. The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others. Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs). These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12. In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.
Subject(s)
Chromosomes, Human, Pair 12 , Cosmids/genetics , Gene Library , Sequence Tagged Sites , Base Sequence , Chromosome Mapping , Chromosomes, Artificial, Yeast , DNA/genetics , Genetic Markers , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Polymerase Chain Reaction , Repetitive Sequences, Nucleic AcidABSTRACT
We report the construction of eight human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow-sorting, and the extracted DNA was cleaved with HindIII before cloning into lamba Charon 21A. There is now a complete digest HindIII library containing greater than five chromosome equivalents for each human chromosome. These are available to the scientific community through the American Type Culture Collection in Rockville, MD. The amount of hamster DNA in libraries in which the chromosome was sorted from human x hamster hybrid cells was estimated by species-specific hybridization. It ranged from 5% to 39%. The sorted chromosomes were examined by fluorescence in situ hybridization with species-specific DNA, and the main source of the hamster DNA contamination was found to be intact hamster chromosomes. In addition, we examined a chromosome 21 library, LL21NS02, for clones that fail to grow on the rec+ host LE392. Less than 0.6% of the recombinant phage exhibited the rec+-inhibited phenotype.
Subject(s)
DNA , Gene Library , Animals , Cell Line , Chromosomes, Human, Pair 21 , Cloning, Molecular , Cricetinae , DNA/genetics , DNA/isolation & purification , Deoxyribonuclease HindIII , Escherichia coli/genetics , Humans , Hybrid Cells , Nucleic Acid Hybridization , Recombination, Genetic , Species SpecificityABSTRACT
Several strains of tobacco ringspot virus (TobRV) support the replication and encapsidation of satellite tobacco ringspot virus RNA (STobRV RNA). We have compared the nucleotide sequences of four STobRV RNAs, each initially associated with a different isolate of TobRV. A STobRV RNA from a geranium isolate of TobRV and STobRV RNA from the previously analyzed budblight isolate (J.M. Buzayan, W.L. Gerlach, G. Bruening, P. Keese, and A.R. Gould, 1986, Virology 151, 186-199) differed by a single nucleotide residue substitution. STobRV RNAs from TobRV isolates 62L and NC-87 have the same 360-residue nucleotide sequence. This sequence differs from that of the 359-nucleotide residue budblight STobRV RNA principally at locations 100 through 140. The differences between the two sequences in this region are consistent with a rearrangement of blocks of nucleotide residues. The two sequences can be folded with similar patterns of base pairing. All four STobRV RNAs share a sequence of eighty 5'-terminal and of twenty 3'-terminal residues, including the 5' hydroxyl group and 2':3'-cyclic phosphodiester group.
