Satish Parimoo

Identification of seven new human MHC class I region genes around the HLA-F locus.

Using cDNA hybridization selection techniques, we identified seven new genes in a 280 kilobase YAC coveting theHLA-F locus. The new genes were mapped back to the YAC by a combination of optical restriction mapping and pulse field gel electrophoresis. Northern analysis of individual clones demonstrated the presence of either different mRNA sizes or different expression patterns. Two of the cDNA clones were expressed only in lymphoid cell lines: one in Jurkat cells (T cell) and another in JY cells (B cell). All the genes lacked sequence similarity to any known classical and non-classical major histocompatibility complex (MHC) class I genes, indicating that theMHC class I region has more functions than anticipated. Of the seven new genes, one is highly similar (97%) to mouse 60S ribosomal protein, and another is homologous to diubiquitin proteins. Of the two G-coupled receptor-like cDNAs, one was fully sequenced and found to be an olfactory receptor-like gene. The study strengthens evidence that theMHC complex not only plays a key role in the immune system, but also contributes to non-immunological functions.

Construction and characterization of a Notl-BsuE linking library from the human X chromosome

Abstract We describe the construction and characterization of methylation-resistant sequence-tagged Not I linking clones specific for the X chromosome, referred to as Not I- Bsu E linking clones. The approach consists of methylating the X-chromosome-specific cloned DNA with Bsu E methylase (M. Bsu E), an enzyme that methylates the first C residue in the CGCG sequence, followed by selection of the methylation-resistant Not I sites by insertion of a kanamycin-resistance gene in the clones cleavable by Not I. The frequent occurrence of Not I sites in CpG islands is expected to cause methylation of a large number of Not I sites with Bsu E methylase, thereby rendering them resistant to Not I cleavage. Thus, the combination of M. Bsu E and Not I yields less frequent cutting than the Not I alone. We have isolated, partially squenced, and characterized 113 Not I- Bsu E linking clones, and mapped 50 clones to various regions along the chromosome.

CDNA SELECTION : AN APPROACH FOR ISOLATION OF CHROMOSOME-SPECIFIC CDNAS

Publisher Summary This chapter details the simple, membrane-based cDNA selection approach with random primed cDNA libraries and the results obtained in its application in the human HLA locus. The cDNA selection approach is a relatively simple, efficient, and rapid method of identification of chromosome- or subchromosome-specific cDNAs. The approach is certainly more efficient and sensitive in detecting rare species of cDNAs than is direct screening of full-length cDNA libraries with a series of yeast artificial chromosomes (YACs). Many disease genes have been identified using either cDNA selection or exon amplification methods. Although any particular method is not without limitations, the decision to use a particular methodology for identifying genes is largely dependent on the availability of starting materials and the ultimate goal. Hence, a particular situation may even demand the use of more than one approach to complement the limitations of the others, until the time when the human genome is completely sequenced, cDNA selection will remain one of the simple and reliable methods for identification of cDNAs from large genomic segments.

Locus Specific Identification of Transcribed Sequences Using Yacs and Whole Yeast Genomic DNA

A large fraction of mammalian genomes contain DNA which appears to be very poor in biologic information. Unless or until nucleic acid sequencing procedures emerge that are capable of a much higher throughput of good quality sequence at a lower cost than the present protocols, it appears attractive to approach characterization of these genomes by focussing attention on transcribed regions and immediately adjacent sequences. The advantages gained by such an approach are that nearly all the genes of man will be identified and probes for these genes made available long before the first human genome is sequenced. Our laboratory has been concentrating on hybridization selection approaches (1) to identify transcribed sequences in yeast artificial chromosomes (YACs) or other cloned genomic DNA, and on affinity selection procedures to identify cDNAs that embed particular motifs.