Annie Hampe

Nucleotide sequence of HIV1-NDK: a highly cytopathic strain of the human immunodeficiency virus

A highly cytopathic strain of HIV1, named HIV1-NDK, has been isolated from a Zaïrian patient affected with AIDS. This isolate is 10(4) times more cytopathic and infectious than the prototype. To correlate the high cytopathic properties of this strain with genetic variations, we have cloned and sequenced the genome of this isolate. The principal feature which could be drawn from the fine analysis of the HIV1-NDK sequence is that the variability is not clustered in one particular region but rather spread out all along the genome. Only minor differences seem to be responsible for the acute biological effect of HIV1-NDK.

A 356-Kb Sequence of the Subtelomeric Part of the MHC Class I Region

The subtelomeric part of the MHC Class I region contains 11 of the 21 genes described on chromosome 6 at position 6p21.3. The general organization of those and other genes resident in the region was revealed by determining a 356,376 bp sequence. Potential exons for new genes were identified by computer analysis and a large number of ESTs were selected by testing the sequence by the BLAST algorithm against the GenBank nonredundant and EST databases. Most of the ESTs are clustered in two regions. In contrast, the whole HLA-gene region is crammed with LINE and SINE repeats, fragments of genes and microsatellites, which tends to hinder the identification of new genes.

Transcriptional analysis of the 69-kb sequence centromeric to HLA-J: a dense and complex structure of five genes.

Abstract. Performed within the framework of the sequencing of the 356-kb MHC class I distal region, systematic bioinformatic annotation and preliminary experiments conducted on the whole sequence indicate a high level and a complex pattern of expression. In this paper, we analyze a particular stretch of 69 kb centromeric to the HLA-J gene, in which we identify 21 different mRNAs mainly expressed in testis, and characterize five different transcription units, HZFw, HZFc, HCGV, HTEX6, and HTEX4. These tightly linked genes form a cluster conserved between human and mouse and displaying a high gene density of about one every 14 kb. Alternative splicing processes are observed for all the genes, together with an alternative polyadenylation event for gene HTEX4, sense/antisense mRNA overlaps for HZFw and HZFc, for HZFw and HCGV at their 3′ end, and for HTEX6 and HTEX4 at their 5′ end. This complex genomic structure suggests a mechanism of coregulation by cis-interaction in gene expression.

A NEW NON-HLA MULTIGENE FAMILY ASSOCIATED WITH THE PERB11 FAMILY WITHIN THE MHC CLASS I REGION

In an effort to initiate steps designed to characterize the idiopathic hemochromatosis disease gene, theHLA-A/HLA-F region where this gene is in disequilibrium linkage with some polymorphic markers has been over-lapped by a yeast artificial chromosome (YAC) contig. In order to achieve the physical mapping of these YACs and of the corresponding genomic region, we subcloned one of the YACs involved. A computer-assisted analysis of the sequence of one subclone led to the isolation of a potential exon that proved to belong to a new expressed messenger namedHCGIX. After southern blot analysis, the corre-sponding cDNA clone was found to belong to a new multigene family whose members are dispersed throughout theHLA class I region and are closely associated with members of another recently described multigene family designatedPERB11. The data reported here suggest that these two multigene families form a cluster that have been dispersed together throughout the telomeric part of the major histocompatibility complex and have been involved in the genesis of this human class I region.

HTEX4, a new human gene in the MHC class I region, undergoes alternative splicing and polyadenylation processes in testis

In the course of our effort to systematically sequence a 400 kilobase (kb) region centromeric to HLA-F, we previously reported the 37 kb sequence of cosmid 13B. This short area contains at least four new computer-predicted genes, organized in a clustered structure which also includes gene HCG-V, the human orthologue of the mouse tctex5 gene (Giffon et al. 1996; Lepourcelet et al. 1996). One of these genes, HTEX4, produces at least five transcripts detected by a genomic probe (probe 6) containing the GRAIL-predicted exon 271 (Fig. 1; Lepourcelet et al. 1996). One HTEX4 mRNA (7.8 kb) is ubiquitously expressed at a low level, two are specifically expressed in colon (3 kb and 1.7 kb), and two correspond to a testis-specific expression (1.5 kb and 4.1 kb). Accordingly, screening of a human testis cDNA library (Stratagene, La Jolla, Calif.) with probe 6 led to the identification of three different clones. Two of these, designated 1 and 5, contained a polyA+ tail at the 39 end. The third clone, designated 6, resulted from a random primed cDNA. Its 39 end was obtained by a hemi-nested polymerase chain reaction (PCR) on the testis cDNA library, using the universal M13 primer (Pu) and successively oligonucleotides 6U and 16 (Table 1). Clone 1, 5002 base pairs (bp), corresponds to a single exon most probably defining the 39 end of the 7.8 kb messenger (Fig. 1 A). A 9-nt sequence, GTGGTGGCA, located 723 bp upstream of the polyA+ tail and within an Alu repeated sequence, was not found in the corresponding genomic sequence, suggesting the occurrence of a length polymorphism ± probe 6 indeed corresponds to a singlecopy probe (data not shown) and clone 1 sequence displays no redundancy elsewhere in the 400 kb sequence centromeric to HLA-F (A. Hampe, personal communication). The 1703 bp sequence of clone 5 defines four exons of perfect match with the genomic sequence (Fig. 1 A). A distal intron of 21118 bp places the last exon, 523 bp, centromeric to the 39 end of the HCG-VII gene within the telomerically adjacent cosmid 3B (Lepourcelet et al. 1996). Clone 6 sequence (1137 pb) defines three exons of 100% identity with the cosmid 13B sequence (Fig. 1 A). The last exon also results from an alternative polyadenylation event. Clones 5 and 6, but not 1, share a short exon of 76 bp followed by an identical 59 splicing site at the next exon. However, this common 76 bp exon is preceded in each mRNA by a different exon. By probing northern blots with an exon-284 PCR product, named 6U-6L, clone 6 was found to correspond to the shorter messenger, 1.5 kb in length (Fig. 1B). Clone 6 thus lacks its 59 end some 300 bases at the most. Probe 5UThe nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AF032109, AF032110, and AF032111

Small deletion in v-src SH3 domain of a transformation defective mutant of rous sarcoma virus restores wild type transforming properties

RSV mutant virus PA101T was obtained while assaying the tumorigenicity of parental PA101 virus in chickens. PA101 is a transformation defective mutant of RSV which has a low src kinase activity. However, PA101 retained a temperature-sensitive ability to induce sustained proliferation of neuroretina cells. PA101T appeared as a wild-type phenotype revertant of PA101. Molecular cloning and sequencing of PA101T showed that this reversion is due to additional mutations in PA101 src gene. These mutations are a deletion eliminating three amino acids in the N-terminal region of SH3 domain and mutation of Ala 426 to Val. Analysis of the properties of chimeric src genes associating either half of PA101T with the complementary regions of PA101 or wild-type virus showed that the N-terminal moiety of PA101T src, which contains the deletion, confers wild-type transforming properties, whereas its C-terminal moiety, which contains single amino acid mutation, confers a partially temperature-sensitive phenotype. These results are consistent with other reports showing that mutations or deletions in this region of SH3 activate the transforming potential of c-src. They support the hypothesis that the N-terminal region of SH3 interacts with a cellular negative regulator of src activity.

A YAC CONTIG AND AN STS MAP SPANNING AT LEAST 3.9 MEGABASEPAIRS TELOMERIC TO HLA-A

Genetic hemochromatosis (GH) is a common autosomal recessive disorder of iron metabolism characterized by excessive absorption of dietary iron through the duodenal mucosa (see Simon and Brissot 1988). Genetic studies have shown that the hemochromatosis gene (HFE) maps to 6p21.3 within a region limited byHLA-F and D6S299 [250 kilobases (kb) and 4 cM telomeric to HLA-A, respectively) (Jazwinska et al. 1993; Calandro et al. 1995; Crawford et al. 1995; Raha-Chowdhury et al. 1995a, b; Gandon et al. 1996)]. When our work was initiated, better physical characterization of the critical region telomerically adjacent toHLA-Awas required for generating and mapping polymorphic markers with a view to refining the location of HFE. This situation prompted us to establish a physical map of this candidate region at least 4 cM in size by means of YAC clones and STS content map. YAC databases were searched for clones positive for the following loci, previously mapped within the HFE candidate region:HLA-F, the myelin/oligodendrocyte glycoprotein (MOG) gene,D6S131E, the ret finger protein ( RFP) gene,D6S306, D6S105,andD6S464(see Figure 1; Gyapay et al. 1994; Volz et al. 1994; Amadou et al. 1995). This search was performed using an html browser on the Baylor College of Medicine (BCM) server (YAC data searches, http://gc.bcm.tmc.edu:8088/bio/yac_search.html), which includes the CEPH-GENETHON, BCM, and MIT databases. We could thus select 24 thoroughly positive YACs from the Centre d’Etude du Polymorphisme Humain (CEPH) library (Table 1). Another 9 STSs previously amplified within some of these YACs were subsequently identified by database searches on the BCM server (Fig. 1). The YACs wereEcoRIandTaqIdigested, blotted, and probed with bothAlu and LINE repetitive elements. The Alu probe was a 300 base pair (bp) restriction genomic fragment belonging to the class III Alu family (Eladari et al. 1992). TheLINE probe was a 1529 bp genomic fragment exhibiting more than 80% identity with human Line-1 repeat mRNA (P. Bouric, personal communication). YAC clones were grouped depending on their Alu and LINE restriction fingerprint similarity. Some YACs were chosen or end fragment amplification using a vectorette polymerse chain reaction (PCR) procedure (see legend to Figure 1). Insert end amplification products were α32P-dCTP radiolabelled with the Rediprime DNA labelling system (Amersham, Buckinghamshire, UK) and hybridized to immobilized human genomic DNA, DNA from human chromosome 6-rodent cell hybrid line GM10629A (from the Human Genetic Mutant Cell Repository, Camden, NJ), yeast DNA from the wild-type strain FY1679, and YAC DNA. Only end probes hybridizing to identical restriction fragments of genomic and chromosome 6 DNAs were used in subsequent analyses. Most YAC insert end fragments were sequenced. Systematic nucleotidic database searches using the BLAST algorithm (Altschul et al. 1990) did not reveal any significant identity, except for 985L (left end of YAC 985A9) which showed 100% identity with the HLA16 sequence. Based on the sequence data of the YAC end inserts, primer sets were developed for specific DNA amplification, resulting in the generation of 13 new STSs. The YACs were then organized by STS content mapping in two preliminary YAC contigs, corresponding to 11 centromeric YACs from 960H11 to 849F12, and to 15 YACs between clones 130F10 and 950H11 (Fig. 1). To fill the remaining gap, two end probes mapped to each side of the telomeric contig ( 130Rand753R; Fig. 1) were used to screen the CEPH (Chumakov et al. 1992), ICI (Anand et al. 1990), and Imperial Cancer Research Fund (ICRF) reference (Lehrach et al. 1990) libraries. Centromeric probe130R identified five clones validated as thorThe nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U50872 ( 10L), U50873 (960R), U50874 (906L), U50875 (985L), U50876 (814L), U50877 (829L), U50878 (906R), U50879 (985R), U50880 (814R), U50881 (17L), U50882 (130R), U50883 (950L), U50884 (306R), and U50885 ( 950R)

Nucleotide sequence analysis of the LTRs and env genes of SM-FeSV and GA-FeSV

The nucleotide sequences of the env genes and the LTRs of SM- and GA-FeSV lambda recombinants have been determined by the Maxam and Gilbert method and/or the dideoxy method with specific sequencing primers. Comparison of the two sequences reveals a homology of 93%, the differences being randomly distributed. Two frameshift mutations are observed in the GA-FeSV isolate which close the reading frame and would prevent the synthesis of the env protein. Comparison of these two FeSV sequences with the env sequences of each antigenic subgroup of FeLV (A, B, C) reveals that these two viruses can be assigned to the A/C subgroups.

Nucleotide sequence study of mouse 5.8S ribosomal RNA

The primary structure of 5.8S mouse ribosomal RNA has been studied and compared to the structures previously established for other animal species. The results obtained show that mouse 5.8S ribosomal RNA yields pancreatic oligonucleotides with the same nucleotide sequence as the homologous oligonucleotides from rat cells. Furthermore T1 oligonucleotides of 5.8S ribosomal RNA from rat, mouse and human cells behave identically on fingerprinting fractionation and have the same composition as judged by pancreatic digestion. These results strongly suggest that the primary structures of 5.8S ribosomal RNA from rat, mouse and human cells are identical. This identity of structure is also found when the presence of several modified bases (psi and methylated bases) is considered. The findings emphasize the remarkable evolutionary stability of ribosomal gene structure. Comparison of the terminal regional of 5.8S RNA with those of 18S RNA reveals differences which imply a more complex mechanism underlying the maturation of 45S precursor RNA than the finding of identical structure would have suggested.

Analysis of large specific T1 oligonucleotides of 17S and 25S ribosomal RNAs from Saccharomyces cerevisiae.

The primary structure of 17S and 25S ribosomal RNAs from Saccharomyces cerevisiae has been analysed by two-dimensional fractionation of T1 oligonucleotides. This method consists of an electrophoresis at pH 3.5 followed by a homochromatography on DEAE-cellulose plates. After the second dimension, the large T1 oligonucleotides were hydrolyzed by pancreatic RNAse, followed by alkaline hydrolysis of the pancreatic products. By fractionating a mixture of tritiated HeLa cell ribosomal RNAs and 32 P yeast cell ribosomal RNAs, two autoradiographs were obtained; one corresponding to the 32P labelled material and the other to the tritiated labelled material. By superposition of the two autoradiographs, the mobility of the various T1 oligonucleotides can be accurately compared and it is shown that yeast 17S rRNA and human 18S rRNA have in common 5 large oligonucleotides and that yeast 25S rRNA and human 28S rRNA have 4 identical oligonucleotides.