Philippe Sanseau

A testis-expressed Zn finger gene (ZNF76) in human 6p21.3 centromeric to the MHC is closely linked to the human homolog of the t-complex gene tcp-11

A novel testis-expressed Zn finger gene (ZNF76) was identified by screening cDNA libraries with cosmids derived from 6p21. ZNF76 is a member of the GLI-Krüppel family of DNA binding proteins. It is conserved in mouse where transcription in testis is initiated at Day 20 after birth. The mouse tcp-11 gene is located in the distal inversion of the t-complex and is developmentally regulated in the same manner as ZNF76. The human homolog of tcp-11 was isolated to allow a precise chromosomal localization. By using a combination of somatic cell hybrids, radiation hybrids, metaphase and interphase fluorescent in situ hybridization, and pulsed-field gel electrophoresis, we mapped the two genes to the 6p21.2 to 6p21.3 region and linked them to each other within 300 kb of DNA, approximately 2 Mb centromeric to the major histocompatibility complex.

Cloning and characterization of human phosphatase inhibitor-2 (IPP-2) sequences

AbstractcDNA clones similar to rabbit muscle phosphatase inhibitor-2 (IPP-2) were isolated from human libraries. On Northern blots two transcripts of ∼2kbp and ∼4kbp were detected in all tissues tested. Analysis of cDNA sequences showed that the longer transcripts were similar to the shorter clones but contained extended 3′ ends. The human nucleotide sequence was highly homologous (94% identity) to the rabbit IPP-2 sequence and encoded a peptide of 205 amino acids. IPP-2 sequences were highly conserved throughout vertebrates. Southern hybridization results were consistent with the existence of a family of related IPP-2 sequences in the human genome. Most of these are likely to be pseudogenes, since all of the cDNA clones examined could have originated from a single gene. By in situ hybridization IPP-2 sequences were mapped to several different human chromosomes. We sequenced one gene located in the major histocompatibility complex (MHC) on Chromosome (Chr) 6 that contained the entire coding region of IPP-2.

Efficiency and specificity of gene isolation by exon amplification

Exon amplification is an increasingly popular approach to the identification of transcribed sequences and will complement other strategies to isolate genes. We have used this system to amplify candidate exons from 32 cosmids, including 8 cosmids which span a well characterized 185-kb region of the human major histocompatibility class II region on Chromosome (Chr) 6. We have examined the efficiency, specificity, and reproducibility of the system in isolating exons from genes known to be present on particular cosmids and have determined the nature and frequency of artefact amplifications in routine cosmid screening. We were able to clone at least one exon from 88% (7/8) of all known genes tested (including exons which are differentially spliced) and obtained artefacts from 19% (6/32) of the cosmids tested. Such artefacts generally arise from the amplification of noncoding sequences flanked by regions with high homology to acceptor and donor splice junctions. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids which have different numbers of genes and gene structures and describe several approaches to the characterization of novel exons cloned in this study.

DNA Sequencing of the MHC Class II Region and the Chromosome 6 Sequencing Effort at the Sanger Centre

The human Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6 (6p21.3) and spans about 4 Mb. According to different gene families the MHC is subdivided into a class I, class II and class III region and many of its gene products are associated with the immune system and the susceptibility to various diseases. To date, we have sequenced about 40% (400 kb) of the class II region between HLA-DP and HLA-DQ and a coordinated effort to sequence the entire MHC is well underway. Analysis of the sequence revealed several novel genes and provides new insights into the molecular organisation and evolution of the MHC. All our data are publicly available via the MHC database (MHCDB) which allows rapid access, retrieval and display in the context of other MHC associated data. MHCDB is online available at (http:(/)/www.hgmp.mrc.ac.uk/) and, together with all our sequences also via anonymous ftp (ftp.icnet.uk/icrf-public).

Cloning of the region between HLA-DMB and LMP2 in the human major histocompatibility complex

The human MHC is one of the most extensively mapped regions of the human genome. Almost all of the class II region of the MHC has already been cloned in cosmids but a gap remained between the DMB and LMP2 genes. Previously, screening of several complete cosmid libraries had failed to bridge this gap, which may contain novel antigen processing or presentation genes. We constructed cosmid libraries from two different sources in order to clone the region: (a) a library with fourfold coverage made from flow-sorted human chromosome 6 DNA and (b) a library derived from a yeast artificial chromosome clone spanning the region. Using this saturation approach, cosmid clones were eventually isolated over the region of interest. A single bacteriophage P1 clone was also obtained spanning the region. The YAC, cosmid, and P1 physical maps were consistent and the distance between the DMB and LMP2 genes was measured as 70 kb. It is not clear why DMB to LMP2 is infrequently represented in cosmid libraries, but the clones that we have obtained will now enable us to search for new coding sequences.

Organisation and Functions of Class II Genes and Molecules

The class II region of the human MHC contains all of the known class II genes: as well as antigen processing components and only one gene not obviously associated with the immune system, RING3. As an approach to understanding linkage disequilibrium and recombination in relation to polymorphism of the region we are cloning and sequencing the class II region. To date, the sequence of the DP-DQ region has almost been completed (see Report by S. Beck). Several sets of genes implicated in the immune system, especially in antigen processing and presentation, are clustered together in the MHC: class I (HLA-A, B, C etc) class II (DR, DQ, DP, DN, DO, DM) LMP2 and 7, TAP1 and 2, TNF, C2, C4, Bf, Hsp70. This situation has provoked speculation that the MHC behaves as a gene cluster in which allelic products of polymorphic genes are maintained on a haplotype so as to co-ordinate T cell repertoire development and deployment. The high levels of linkage disequilibrium across the region are consistent with this idea. Functions of the genes in the MHC are being investigated as a step towards gaining insight into antigen processing and presentation as well as understanding MHC-disease associations. We are concentrating on the functions of the class II-related genes, DM and DN/DO as well as the TAP/LMP cluster.