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DNA octamer element can confer E1A trans-activation, and adenovirus infection results in a stimulation of the DNA-binding activity of OTF-1/NFIII factor.

Adenovirus E1A-dependent trans-activation of viral transcription involves the utilization and alteration of multiple sequence-specific transcription factors. Cellular genes are also activated by E1A, one example being the immunoglobulin heavy-chain locus when assayed by transfection into fibroblast cells. We have explored the basis for the E1A-dependent activation of this cellular transcription unit. We demonstrate that the ATTTGCAT ("octamer") element found in the heavy-chain enhancer and promoter is a target for E1A trans-activation since this sequence can confer inducibility to the normally unresponsive simian virus 40 early promoter. In addition, adenovirus infection stimulates the DNA-binding activity of the ubiquitous octamer-specific factor, OTF-1, and we presume that this is the basis for the stimulation of transcription. Although there are no octamer elements in the adenovirus genome that are known to be important for transcription, there are octamer elements in the viral terminal repeat sequences. These elements bind the NFIII factor and are important for the initiation of DNA replication. Since the NFIII factor has been shown to be identical to OTF-1, we suggest that the stimulation of OTF-1/NFIII activity during an adenovirus infection may be important for efficient initiation of adenovirus DNA synthesis.

Adenovirus Early Proteins↗

His1 and His2 are distantly related, spindle-shaped haloviruses belonging to the novel virus group, Salterprovirus.

Spindle-shaped viruses are a dominant morphotype in hypersaline waters but their molecular characteristics and their relationship to other archaeal viruses have not been determined. Here, we describe the isolation, characteristics and genome sequence of His2, a spindle-shaped halovirus, and compare it to the previously reported halovirus His1. Their particle dimensions, host-ranges and buoyant densities were found to be similar but they differed in their stabilities to raised temperature, low salinity and chloroform. The genomes of both viruses were linear dsDNA, of similar size (His1, 14,464 bp; His2, 16,067 bp) and mol% G+C (approximately 40%), with long, inverted terminal repeat sequences. The genomic termini of both viruses are likely to possess bound proteins. They shared little nucleotide similarity and, except for their putative DNA polymerase ORFs, no significant similarity at the predicted protein level. A few of the 35 predicted ORFs of both viruses showed significant matches to sequences in GenBank, and these were always to proteins of haloarchaea. Their DNA polymerases showed 42% aa identity, and belonged to the type B group of replicases that use protein-priming. Purified His2 particles were composed of four main proteins (62, 36, 28 and 21 kDa) and the gene for the major capsid protein was identified. Hypothetical proteins similar to His2 VP1 are present in four haloarchaeal genomes but are not part of complete prophages. This, and other evidence, suggests a high frequency of recombination between haloviruses and their hosts. His1 and His2 are unlike fuselloviruses and have been placed in a new virus group, Salterprovirus.

Amino Acid Sequence↗

The Anopheles albimanus white gene: molecular characterization of the gene and a spontaneous white gene mutation.

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus.

ATP-Binding Cassette Transporters↗

Emergence of two types of nondechlorinating variants in the tetrachloroethene-halorespiring Desulfitobacterium sp. strain Y51.

Desulfitobacterium sp. strain Y51 exhibits a strong dechlorinating activity for tetrachloroethene (PCE), converting it to cis-1,2-dichloroethene via trichloroethene by the action of the PceA reductive dehalogenase (encoded by pceA). The gene organization around the pceA gene cluster was determined to be in the following order: orf4, orf3, ISDesp1, pceA-B-C-T-mcpA, and ISDesp2, where the pceA gene cluster is surrounded by two nearly identical copies of the ISDesp insertion sequence. Serial subculture of strain Y51 gave rise to variants that abolished the PCE-dechlorination activity. Southern hybridization analysis revealed two types of variants termed small deletion (SD) and large deletion (LD). The characterization of both variants revealed a genetic rearrangement around the pceAB gene cluster. In variant SD, ISDesp1 comprised of 1,572 bp was deleted, which includes the tnpAa encoding IS256 family transposase and unknown orf1. The ISDesp1 contained the inverted terminal repeat sequence and a -35 promoter stretch just upstream of the pceA gene, indicating that this IS element is involved in the formation of the variant SD. Loss of the pceA transcription changed the variant SD to the PCE-nondechlorinating phenotype. The variant LD lost the 6.5-kb region, including one copy of ISDesp and the pceABCT-mcpA gene cluster, confirming that the homologous recombination is associated with the emergence of this variant.

Base Sequence↗

Tat protein of human immunodeficiency virus type 1 is a monomer when expressed in mammalian cells.

Human immunodeficiency virus type 1 (HIV-1) encodes a transactivator protein, known as Tat, that stimulates transcription directed by the HIV-1 long terminal repeat sequences. Tat appears to bind directly to the TAR RNA element present at the 5' end of nascent HIV-1 transcripts and thereby stimulates the activity of transcription complexes. We have expressed Tat in simian COS cells by transfection of a mammalian expression vector. Using immunoblots to detect Tat, the results of gel filtration and velocity sedimentation analyses demonstrate that Tat is a monomer in COS cell extracts. These results agree with other studies which indicate that Tat is a monomeric protein.

Animals↗

Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase.

Triple tandemly repeated sequences and the corresponding complementary sequence are known to exist in the 5'-terminal regulatory region of the human gene for thymidylate synthase (TS). To examine the function of these sequences, a set of deletion mutants was prepared and used in a transient expression assay. The results showed that at least one repeated sequence and its complementary sequence were necessary for the efficient expression of the gene. As another approach to understanding the function of this unique structure, DNA polymorphism in the same region was analyzed. In addition to the TS gene with the triple tandem repeat, the TS gene with a double tandem repeat was found in genomes of normal human subjects at an estimated frequency of 19% when genomes of 21 unrelated Japanese were analyzed. The expression activity of a reporter gene linked to the promoter region of the human TS genes with the two types of repeated sequence was examined and the result showed that the expression activity of the gene with the double repeat was lower than that of the gene with the triple repeat in the transient expression assay. Thus, it appears that the unique repeated sequences in the 5'-terminal region of the human TS gene are polymorphic and contribute to the efficiency of expression of the gene.

Base Sequence↗

The fugitive LTR retrotransposon from the genome of the human blood fluke, Schistosoma mansoni.

Inspection of the nucleotide sequence of bacterial artificial chromosome number 49_J_14 [Le Paslier, M.C., Pierce, R.J., Merlin, F., Hirai, H., Wu, W., Williams, D.L., Johnston, D., LoVerde, P.T., Le Paslier, D., 2000. Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library. Genomics 65, 87-94] from chromosome 1 of the genome of Schistosoma mansoni (GenBank ) revealed the likely presence of a proviral form of a novel schistosome retrotransposon. The novel element, which we named the fugitive, belonged to the mag-like family of the gypsy-Ty3 clade of long terminal repeat retrotransposons. It was closely related to the mag-like retrotransposon Gulliver from Schistosoma japonicum, but was dissimilar to several other long terminal repeat retrotransposons known from S. mansoni including Boudicca, Saci-1, Saci-2 and Saci-3. The full length fugitive element was 4811 bp constituted of a single read-through open reading frame of 4134 bp flanked at both ends by identical long terminal repeat sequences of 273 bp. The open reading frame encoded retroviral-like gag, with a distinctive double Cys-His motif, and pol polyprotein, with a pol domain order of protease, reverse transcriptase, RNaseH and integrase. Examination of schistosome transcriptome sequences in the public domain revealed that the fugitive was transcribed in at least six developmental stages of S. mansoni, while bioinformatics approaches and Southern hybridisation analysis indicated that as many as 2000 copies of the fugitive were interspersed throughout the schistosome genome.

Amino Acid Sequence↗

Phenotypic reversion of an IS1-mediated deletion mutation: a combined role for point mutations and deletions in transposon evolution.

We have physically characterised a deletion mutant of the R plasmid R100 which has lost all of the antibiotic resistances, including chloramphenicol resistance (Cmr), coded by its IS1-flanked r-determinant. The deletion was mediated by one of the flanking IS1 elements and terminates within the carboxyl terminus of the Cmr gene. DNA sequence analysis showed that the mutated gene would produce a protein 20 amino acids longer than the wild-type due to fusion with an open reading frame in the IS element. Surprisingly for a deletion mutation, rare, spontaneous Cmr revertants could be recovered. Two of the four revertants studied had frame shifts due to the insertion of a single AT base pair at the same position; the revertants would code for a protein five amino acids shorter than the wild-type. The other two revertants had acquired duplications of the 34-bp inverted terminal repeat sequences of the IS1 element and would direct the synthesis of a protein six amino acids longer than the wild-type. The reverted Cmr markers were still capable of transposition. These observations suggest a role for point mutations and small DNA rearrangements in the formation of new gene organisations produced by mobile genetic elements.

Base Sequence↗

Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes.

Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.

Cell Line↗

Exchange of genetic sequences of long terminal repeat and the env gene by a promiscuous primate type D retrovirus.

Squirrel monkey retrovirus (SMRV) is a New World primate type D retrovirus. It was shown that SMRV-related sequences could be detected in another New World species, the skunk. It was further suggested that SMRV and an Old World primate type C retrovirus, baboon endogenous virus (BaEV), may have exchanged their env gene sequences. In this study, we sought to understand which sequences were exchanged between the genomic DNAs of SMRV and skunk. We also sought to determine the sequences exchanged between SMRV and BaEV. Here, we demonstrate that the long terminal repeat of SMRV is present in the skunk genome. We also show, by nucleotide sequence analysis, that the env gene that encodes the p15E glycoprotein of BaEV was most likely transduced from the corresponding gene of a primate type D retrovirus. Our results demonstrate that SMRV is a promiscuous virus with its pol gene homologous to the pol genes of type A, type B and avian type C viruses and a portion of its env gene homologous to the env genes of primate type C retroviruses. However, the primer binding sequence is unique to type D retroviruses. These kinds of recombination are likely to occur more than once in the evolution of retroviruses. The promiscuous nature of retroviruses and the recent incidence of unintended retroviral integration into a gene therapy patient underscore the importance of understanding how retroviral sequences are recombined among themselves and how they are integrated into the mammalian genome.

Amino Acid Sequence↗

Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization.

We have used differential cell extraction and conventional chromatography to separate and partially purify the four adeno-associated virus (AAV) nonstructural proteins Rep78, Rep68, Rep52, and Rep40. In the cytoplasmic extracts Rep52 and Rep40 were present in greater abundance than Rep68 and Rep78, with Rep78 being the least abundant. In nuclear extracts the four Rep proteins were approximately equal in abundance. Regardless of the subcellular fraction examined, three of the Rep proteins (Rep78, Rep68, and Rep40) consisted of two protein species with slightly different mobilities during polyacrylamide gel electrophoresis. In contrast, Rep52 consisted of only one protein species. Both Rep78 and Rep68 were capable of binding efficiently to AAV terminal hairpin DNA substrates, but we could not detect site-specific DNA binding by Rep52 and Rep40. Like Rep68, Rep78 had both an ATP-dependent trs endonuclease and a DNA helicase activity. Both Rep78 and Rep68 cut the terminal AAV sequence at the same site (nucleotide 124). The binding, trs endonuclease, and DNA helicase activities comigrated during sucrose density gradient centrifugation with a mobility expected for a monomer of the protein, suggesting that the three biochemical activities were intrinsic properties of the larger Rep proteins. The chromatographic behavior and the DNA-binding properties of the four Rep proteins identified at least two domains within the rep coding region, an exposed hydrophobic domain within the C-terminal end (amino acids 578 to 621) and a region within the N terminus (amino acids 1 to 214) which was necessary for binding to the terminal repeat sequence. No site-specific nuclease activity was seen in the presence of nucleotide analogs ATP-gamma-S or AMP-PNP, suggesting that ATP hydrolysis was required for the endonuclease reaction. Furthermore, although ATP was the only cofactor which would support the trs endonuclease activity of Rep78, Rep68 nuclease activity was seen in the presence of several other nucleotide cofactors, including CTP, GTP, and UTP.

Binding Sites↗

Capture of DNA sequences at double-strand breaks in mammalian chromosomes.

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.

Animals↗

Mutations in the founder of the MIP gene family underlie cataract development in the mouse.

The major intrinsic protein (MIP) of the vertebrate eye lens is the first identified member of a sequence-related family of cell-membrane proteins that appears to have evolved by gene duplication. Several members of the MIP family transport water (aquaporins), glycerol and other small molecules in microbial, plant and animal cells. Mutations in two aquaporin homologues of MIP underlie an autosomal recessive form of nephrogenic diabetes insipidus and absence of the Colton blood group antigens in humans, whereas, mutation of a third MIP-like gene underlies 'big brain' development in Drosophila. Here we show that distinct mutations in the murine Mip gene underlie one form of autosomal dominant cataract in the mouse. The cataract Fraser mutation is a transposon-induced splicing error that substitutes a long terminal repeat sequence for the carboxy-terminus of MIP. The lens opacity mutation is an amino-acid substitution that inhibits targeting of MIP to the cell-membrane. These allelic cataract mutations provide the first direct evidence that MIP plays a crucial role in the development of a transparent eye lens.

Amino Acid Sequence↗

Genetics and expression of the Q6 and Q8 genes. An LTR-like sequence in the 3' untranslated region.

A unique 2.2-kb mRNA is transcribed from the Q6 and Q8 genes of the mouse major histocompatibility complex. RNase protection experiments and DNA sequence analysis have mapped the 3' terminus to a site located 1110 bp downstream from exon 8. Comparison of the 3' sequence of Q8 to that of Q7 revealed that the two genes diverge from one another at a point located 200 bp into the untranslated domain. This finding explains the increased size of the transcript. RNase protection experiments involving twelve different strains of mice have revealed few sequence polymorphisms. A tissue distribution of the 2.2-kb transcript in B6 mice revealed that the highest quantities of message were present in the spleen with decreasingly lower amounts in the thymus, liver, kidney, testis, and brain. The H-2r haplotype appeared novel; it is phenotypically Qa-2-, yet expressed a 2.2-kb transcript which hybridizes to a Q8 probe. A comparison of a Qa-2hi expressor, B6, and a Qa-2low expressor, BALB/cFla, has established that these quantitative cell surface differences are reflected in mRNA differences. A homology search of the Genbank database has revealed that the 3' portion of Q8 contains extensive homology to a retrovirus-like long terminal repeat sequence that is characteristic of an embryonic-specific transposon (ETn).

Alleles↗

Molecular cloning of the endogenous rat C-type helper virus DNA sequence: structural organization and functional analysis of some restricted DNA fragments.

Recently, we have identified and purified the integrated and proviral DNA sequences specific for two endogenous rat type C leukaemia helper viruses: WR-RaLV which originated from a fibrosarcoma induced in a feral rat and RHHV from the cell line HTC-H1 which originated from a Buffalo rat hepatoma. The rat leukaemia helper virus DNA sequences have previously been shown to be 8.4 to 8.8 kilobases (kb) in size. In this communication, we report the molecular cloning of the 8.8 kb DNA of RHHV by ligation at the BamHI site of the vector pBR322, cultured in an Escherichia coli RR1 host. After screening 5750 clones for ampicillin resistance and tetracycline sensitivity and testing by colony hybridization using 32P-labelled RHHV cDNA, four clones were isolated, two of which carried the total 8.8 kb DNA. A detailed restriction endonuclease map of the cloned RHHV DNA was deduced by sequential digestions of either 3'- or 5'-labelled DNA. Of the 14 restriction enzymes tested, EcoRI, BamHI, PstI, KpnI, TaqI, PvuII and SmaI gave informative cleavage patterns. At least two copies of long terminal repeated sequences (LTR) flanking the 3' and 5' termini of the proviral DNA were identified by TaqI and PstI cleavages. LTR in the rat endogenous leukaemia helper virus DNA measured 780 +/- 20 nucleotides in length. The genetic information encoded by the cloned DNA was also analysed by hybridization selection of RHHV mRNA, which was then used in cell-free protein synthesis in a rabbit reticulocyte lysate system. Essentially all major RaLV-specific proteins precipitable by anti-RaLV serum were synthesized in vitro, confirming that the RHHV genomic DNA was successfully cloned with little fidelity loss or scrambling of the genetic information.

Animals↗

Phagocytosis of Mycobacterium tuberculosis modulates human immunodeficiency virus replication in human monocytic cells.

Macrophage activation resulting from phagocytosis has the potential to modulate human immunodeficiency virus (HIV) replication. We have determined the effects of phagocytosis of particulate stimuli on transcription and release of HIV. Using THP-1 and Mono Mac 6 human monocytic cell lines transfected with HIV long terminal repeat sequence chloramphenicol acetyltransferase (LTR CAT) constructs we have demonstrated that phagocytosis of Mycobacterium tuberculosis enhanced HIV-1 and -2 LTR CAT expression. However phagocytosis of zymosan or inert latex beads had little or no effect on CAT expression. Enhancement of HIV LTR CAT expression was dependent upon intact NF-kappa B binding sites and was independent of tumour necrosis factor alpha secretion. M. tuberculosis strains of different degrees of virulence induced similar levels of enhanced CAT expression. In contrast, phagocytosis of M. tuberculosis by HIV-1-infected THP-1 cells reduced supernatant reverse transcriptase (RT) activity without suppression of p24 antigen release. Phagocytosis of zymosan granules or latex particles did not alter released RT activity. However, phagocytosis of either M. tuberculosis, zymosan granules or latex particles by HIV-1-infected human peripheral blood monocyte-derived macrophages reduced supernatant RT activity. These data indicate that phagocytosis of M. tuberculosis may enhance HIV transcription in monocytic cells although it may reduce release of intact HIV.

Antibodies, Monoclonal↗

Retroviral insertions into a herpesvirus are clustered at the junctions of the short repeat and short unique sequences.

We previously described the integration of a nonacute retrovirus, reticuloendotheliosis virus (REV), into the genome of a herpesvirus, Marek disease virus (MDV), following both long-term and short-term coinfection in cultured fibroblasts. The long-term coinfection occurred in the course of attenuating the oncogenicity of the JM strain of MDV and was sustained for > 100 passages. The short-term coinfection, which spanned only 16 passages, was designed to recreate the insertion phenomenon under controlled conditions. We found that REV integrations into MDV were common and could occur within the first passage following coinfection. Now we have mapped the integration sites. After 5-16 passages in vitro, 17 out of 19 REV junction sites are clustered in two 1-kilobase regions at the junctions of the short unique and short repeat region of MDV. In the long-term cocultivation experiment, 6 out of 10 insertions also mapped in this region. In both cases, integrated proviruses are unstable and undergo subsequent recombinative deletion, often leaving a solitary long terminal repeat. The long terminal repeat sequences are, however, stably maintained for many rounds of passaging in vitro. This clustering of insertions presumably is influenced by selection for viable and fast-growing viruses, and occurs in a region of the MDV genome which shows significant size heterogeneity in several strains.

Animals↗

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus.

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.

Animals↗