Expression of the hepatitis delta virus large and small antigens in transgenic mice.

Simultaneous infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) in humans is often associated with severe viral liver disease including fulminant hepatitis. Since HBV is thought to be noncytopathic to the hepatocyte, the enhanced disease severity observed during dual infection has been attributed to either simultaneous immune responses against the two viruses or direct cytotoxic effects of HDV products on the hepatocyte or both. To examine these alternate possibilities, we produced transgenic mice that express the small and large delta antigens (HDAg) in hepatocyte nuclei at levels equal to those observed during natural HDV infection. No biological or histopathological evidence of liver disease was detectable during 18 months of observation, suggesting that neither the large nor small form of HDAg is directly cytopathic to the hepatocyte in vivo.     

In vivo replication of the hamster polyomavirus genome and generation of specific deletions in the process of lymphomagenesis.

Hamster polyomavirus (HaPV) causes lymphomas when injected into newborn hamsters. These tumors are virus-free but accumulate large amounts of deleted extrachromosomal viral genomes. In order to identify the major sites of virus replication in animals, we have monitored the HaPV DNA present in different organs at various times after injection. The data demonstrate that viral replication preferentially occurs in lymphoid organs. Lymphoma-associated viral genomes display specific deletions. PCR analysis shows that such viral genomes are the only variants detectable in infected animals, suggesting that they are generated by a specific cellular mechanism. We have tested the possible role of the lymphoid cell-specific V(D)J recombination activity in the generation of these specific variants. Our results indicate that this mechanism is not solely responsible for the viral genome rearrangement, if involved at all.     

Chromosome 4q35 haplotypes and DNA rearrangements segregating in affected subjects of 19 Italian families with facioscapulohumeral musculatur dystrophy (FSHD)

Four DNA markers on the distal long arm of chromosome 4 have been analyzed for their linkage to facioscapulohumeral muscular dystrophy locus (FSHD) in a series of 16 Italian families. We found that, in two families, the disease is not linked to the 4q35 markers, indicating the presence of genetic heterogeneity among Italian FSHD families. Linkage analysis in the remaining families supports the order cen-D4S171-D4S163-D4S139-D4S810-FSHD-qter, in agreement with the physical map from the literature. EcoRI digestion and hybridization with the distal marker p13E-11 (D4S810) detected DNA rearrangements in the affected members of both sporadic and familial cases of FSHD, with family-specific fragments ranging in size between 15 kb and 28 kb. In three sporadic FSHD cases, the appearance of a new small fragment not present in either parent was clearly associated with the development of FSHD disease. However, in the familial cases analyzed, we observed two recombinations between all four 4q35 markers and the disease locus in apparently normal subjects, leaving open the possibility of nonpenetrance of the FSHD mutation.

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Optimization of transverse alternating field electrophoresis for strain identification of Leuconostoc oenos

Genomic DNA of several strains oof oenological lactic bacteria belonging to the species Lactobacillus plantarum, Leuconostoc oenos and Pediococcus pentosaceus was digested by the rare-cutting endonucleases ApaI and SmaI. The restriction products were separated by transverse alternating field electrophoresis (TAFE). The size of the genome of L. oenos estimated by adding the molecular size of the ApaI fragments was on average 1320 kb. A strong polymorphism was observed between the strains, which could be easily differentiated except for two industrial strains of L. oenos. A simple modification of the TAFE apparatus is proposed to improve the separation of the DNA fragments. Correspondence to: J.-N. Hallet     

Evolutionary study of multigenic families mapping close to the human MHC class I region

Summary During a search for novel coding sequences within the human MHC class I region (chromosome 6p21.3), we found an exon (named B30-2) coding for a 166-amino-acid peptide which is very similar to the C-terminal domain of several coding sequences: human 52-kD Sjögren's syndrome nuclear antigen A/Ro (SS-A/Ro) and ret finger protein (RFP), Xenopus nuclear factor 7 (XNF7), and bovine butyrophilin. The first three of these proteins share similarities over the whole length of the molecule whereas butyrophilin is similar in the C-terminal domain. The N-terminal domain of butyrophilin is similar to rat myelin/oligodendrocyte glycoprotein (MOG) and chicken B blood group system (B-G) protein. These domains are components of a new subfamily of the immunoglobulin superfamily (IgSF). Butyrophilin is thus a mosaic protein composed of the MOG/B-G Ig-like domain and the C-terminal domain of 52-kD SS-A/Ro, RFP, and XNF7 (1330-2-like domain). Moreover, in situ hybridization shows that RFP, butyrophilin, and MOG map to the human chromosome 6p2l.3-6p22 region and are thus close to the MHC class I genes. It is therefore possible that the butyrophilin gene is the product of an exon shuffling event which occurred between ancestors of the RFP and MOG genes. To our knowledge, this is the first example of the colocalization of a chimeric gene and its putative progenitors. Finally, regulatory protein T-lymphocyte 1 (Rpt-1) shares similarities with the N-terminal halves of RFP, 52-kD SS-A/Ro, and XNF7, but not with the B30-2-like domain. We show that the ancestral Rpt-l gene evolved by overprinting. Correspondence to: P. Pontarotti     

Cross-protection against lymphocytic choriomeningitis virus mediated by a CD4+ T-cell clone specific for an envelope glycoprotein epitope of Lassa virus.

Recombinant vaccinia virus expressing the Lassa virus (LV) envelope glycoprotein precursor, V-LSGPC, was used to study the basis of LV-induced cross-protective immunity against the closely related arenavirus lymphocytic choriomeningitis virus (LCMV). C3H/HeJ mice primed with V-LSGPC developed neither circulating antibodies nor CD8+ cytotoxic T cells specific for LCMV, yet they resisted a normally lethal LCMV challenge. Spleen cells from such mice gave a proliferative response to LCMV in vitro that was inhibitable by anti-CD4 antibody. Synthetic peptides corresponding to predicted T-cell sites common to the envelope glycoprotein precursor (GP-C) of LV and that of LCMV were used to map the specificity of the proliferative response to an epitope located between amino acids 403 and 417 of LV GP-C. Several CD4+ T-cell clones specific for the 403-417 peptide were isolated and found to produce gamma interferon in response to both the peptide and LCMV. One of these clones, C9, was selected for further study. C9 lysed I-AK-bearing target cells, and when adoptively transferred to C3H/HeJ mice, it was capable of mediating both a peptide-specific delayed hypersensitivity reaction and resistance to lethal LCMV challenge. These collective findings demonstrate, for the first time, that CD4+ T cells can play a major role in arenavirus-specific cross-protective immunity.     

Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes.

The paraoxonase/arylesterase gene is located close to the cystic fibrosis gene on chromosome 7. Human serum contains two paraoxonase/arylesterase allozymes, A and B, which differ in their substrate specificities and kinetic properties. Purified A, AB, and B esterases were digested with trypsin, and the resultant peptides were compared by high-performance liquid chromatography. The elution profiles were very similar for all three samples, except for (1) one peptide (i.e., peptide A) seen only in the A and AB profiles and (2) another peptide (i.e., peptide B) seen only in the B and AB profiles. Sequencing revealed that peptide A had glutamine at amino acid position 191, whereas peptide B was generated by cleavage on the carboxy side of position 191, presumably because there was a basic (trypsin-specific) amino acid at that position. Working independently, our laboratory and one other laboratory have sequenced the coding region for paraoxonase from human liver cDNA libraries and have identified two polymorphic sites: Arg/Gln at position 191 and Leu/Met at position 54. Using PCR amplification and direct sequencing of nucleotides in both polymorphic regions with genomic DNA, we have estimated the allelic frequencies and have determined their concordance with the serum paraoxonase allozyme phenotypes in 27 unrelated adults and in 16 members of a three-generation pedigree. Among unrelated individuals, the Met/Leu polymorphism at position 54 did not correlate with the serum esterase phenotype. In contrast, the particular amino acid at position 191 correlated perfectly with serum phenotypes: A-type individuals had Gln at position 191, and B-type individuals had Arg at position 191; AB-type serum was found only with the heterozygous (Arg/Gln) combination. Pedigree analysis showed both polymorphisms to be inherited in the expected Mendelian manner and confirmed that only the 191 polymorphism showed concordance with the serum paraoxonase/arylesterase phenotypes.     

Novel changes in the plasma membrane and cortical cytoplasm during wound-induced contraction in a giant-celled green alga

Summary Changes in the plasma membrane surface and in the cortical cytoplasm during wound healing in giant green algal cells ofErnodesmis verticillata (Kützing) Brgesen were followed using scanning and transmission electron microscopy. Microvillus-like structures that contain cytoplasmic and cytoskeletal constituents were observed emanating from the surface of the plasma membrane at the retracting/cut end of wounded cells. These delicate structures seem to be remnants of cell wall-plasmalemma connections that draw out the plasma membrane and cortical components from the contracting cytoplasm as it pulls away from the cell wall. Most of these connections break during wound healing and, when contraction stops, the microvillus-like protrusions become progressively shorter. In cells treated with a calmodulin antagonist (W-7), a number of distinctive bodies accumulate that are of unknown composition, are oblong in shape, and have a diameter slightly smaller than the protoplasmic protrusions. Ultrastructural and other data indicate that these bodies result from retrieved constituents of the plasma-membrane protrusions, as they do not accumulate in unwounded drugtreated cells or in cells treated in W-5. These findings suggest that the protoplasmic protrusions accumulate membrane and cytoplasmic components that are retrieved and recycled during wound healing inErnodesmis by a novel mechanism. The combined plasma membrane surfaces of the microvillus-like protrusions may help to account for the drastic decrease in surface area that occurs during wound healing.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy - W-7 N-[6-aminohexyl]-5-chloro-1-naph-thalenesulfonamide - W-5 N-[6-aminohexyl]-1-naphthalenesulfonamide     

Chromosomal localization of four human zinc finger cDNAs

cDNA clones encoding zinc finger motifs were isolated by screening human placenta and T-cell (Peer) cDNA libraries with zinc finger (ZNF) consensus sequences. Unique cDNA clones were mapped in the human genome by rodent-human somatic cell hybrid analysis and in some cases in situ chromosomal hybridization. ZNF 80 mapped to 3p12-3qter, ZNF 7 was previously mapped to 8q24 and is here shown by in situ hybridization and use of appropriate hybrids to map telomeric to the MYC locus. ZNF 79 mapped to 9q34 centromeric to the ABL gene and between a constitutional chromosomal translocation on the centromeric side and the CML specific ABL translocation on the telomeric side. ZNF77 mapped to 19p while ZNF 78L1 (pT3) mapped to 19q. Chromosome 19 carries many ZNF loci and other genes with zinc finger encoding motifs; the pT3 clone additionally detected a locus designated ZNF 78L2, which mapped to chromosome region 1p, most likely in the region 1p32 where the MYCL and JUN loci map.