Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli.

The ability to express the genes of pathogenic human viruses, such as the acquired immune deficiency syndrome (AIDS) virus (also called human immunodeficiency virus) in bacterial cells affords the opportunity to study proteins that are ordinarily difficult or inconvenient to obtain in amounts sufficient for detailed analysis. A segment of the AIDS virus pol gene was expressed in Escherichia coli. Expression resulted in the appearance of reverse transcriptase activity in the bacterial cell extracts. The extracts contained two virus-related polypeptides that have the same apparent molecular weights as the two processed forms of virion-derived reverse transcriptase (p66 and p51). The formation of these two polypeptides depended on the coexpression of sequences located near the 5' end of the pol gene, a region that is thought to encode a viral protease. This bacterial system appears to generate mature forms of the AIDS virus reverse transcriptase by a proteolytic pathway equivalent to that which occurs during virus infection of human cells.

The sequence of a large L1Md element reveals a tandemly repeated 5' end and several features found in retrotransposons.

The complete nucleotide sequence of a 6,851-base pair (bp) member of the L1Md repetitive family from a selected random isolate of the BALB/c mouse genome is reported here. Five kilobases of the element contains two overlapping reading frames of 1,137 and 3,900 bp. The entire 3,900-bp frame and the 3' 600 bp of the 1,137-bp frame, when compared with a composite consensus primate L1 sequence, show a ratio of replacement to silent site differences characteristic of protein coding sequences. This more closely defines the protein coding capacity of this repetitive family, which was previously shown to possess a large open reading frame of undetermined extent. The relative organization of the 1,137- and 3,900-bp reading frames, which overlap by 14 bp, bears resemblance to protein-coding, mobile genetic elements. Homology can be found between the amino acid sequence of the 3,900-bp frame and selected domains of several reverse transcriptases. The 5' ends of the two L1Md elements described in this report have multiple copies, 4 2/3 copies and 1 2/3 copy, of a 208-bp direct tandem repeat. The sequence of this 208-bp element differs from the sequence of a previously defined 5' end for an L1Md element, indicating that there are at least two different 5' end motifs for L1Md.

Determination of a functional ancestral sequence and definition of the 5' end of A-type mouse L1 elements.

The complete nucleotide sequence of L1Md-A13, a 6372 base-pair (bp) member of the L1Md repetitive family isolated from a BALB/c mouse genomic DNA library, is reported. The nucleotide sequence of 4331 bp from the 5' end of L1Md-9, which is located in the beta-globin complex of the C57BL/10 mouse, is also reported. Parsimony analysis of these sequences plus two previously reported L1Md sequences allows the determination of an ancestral L1Md sequence. Analysis of the L1Md population indicates that this ancestral sequence is likely to represent a functional L1 sequence. This ancestral sequence confirms that the length (1137 bp and 3900 bp) and relationship (14 bp overlap) of the two large open reading frames previously reported are conserved features of the L1Md family. It also allows the determination of an ancestral amino acid sequence for these two open reading frames. Full-length L1Md elements have one of two sequences tandemly repeated at the 5' end. These two monomers are called A-type and F-type. Our data define the 5' end of A-type full-length L1Md elements. L1Md elements of the A-type have varying numbers of tandemly repeated 208 bp monomers, but each element ends about 78 bp from the 5' end of the terminal 208 bp monomer.

Conservation throughout mammalia and extensive protein-encoding capacity of the highly repeated DNA long interspersed sequence one.

We report an investigation of the structure, evolutionary history, and function of the highly repeated DNA family named Long Interspersed Sequence One (L1). Hybridization studies show, first, that L1 is present throughout marsupial and placental mammalian orders. Second, L1 is more homologous within these species than between them, which suggests that it has undergone concerted evolution within each mammalian lineage. Third, on the whole L1 diverges in accordance with the fossil record. This suggests that it arose in each lineage rather by inheritance from a common ancestral family, which was present in the progenitor to mammals, than by cross-species transmission. Alignment of 1.6 X 10(3) bases of primate and mouse L1 DNA sequences shows a predominance of silent mutations within aligned long open reading frames, indicating that at least this part of L1 has produced functional protein. The observation of additional long open reading frames in further unaligned DNA sequences suggests that a minimum of 3.2 X 10(3) bases or at least half of the L1 structure is a protein-coding sequence. Thus L1, which contains about 100,000 members in mouse, is by far the most repetitive family of which a subset comprises functional protein-encoding genes. The ability of the putative protein-encoding regions of mouse L1 to hybridize to L1 homologs throughout the Mammalia implies that these sequences have been subject to conservative selection upon protein function in all mammalian lineages, rather than in a few. L1 is therefore a highly repeated family of genes with both a widespread and an ancient history of function in mammals.

Nucleotide sequence of the BALB/c mouse beta-globin complex.

The nucleotide sequence of 55,856 base-pairs containing all seven beta-globin homologous structures from chromosome 7 of the BALB/c mouse is reported. This sequence links together previously published sequences of the beta-globin genes, pseudogenes and repetitive elements. Using low stringency computer searches, we found no additional beta-globin homologous sequences, but did find many more long interspersed repetitive sequences (L1) than predicted by hybridization. L1 is a major component of the mouse beta-globin complex with at least 15 elements comprising about 22% of the reported sequence. Most open reading frames greater than 300 base-pairs in the cluster overlap with L1 repeats or globin genes. Polypurine, polypyrimidine and alternating purine/pyrimidine tracts are not evenly dispersed throughout the complex, but they do not appear to be excluded from or restricted to particular regions. Several regions of intergenic homology were detected in dot-plot comparisons of the mouse sequence with itself and with the human beta-globin sequence. The significance of these homologies is unclear, but these regions are candidates for further study in functional assays in erythroid cell lines or transgenic animals.

A directed nucleotide-sequencing approach for single-stranded vectors based on recloning intermediates of a progressive DNA synthesis reaction.

A simple method for site-directed nucleotide sequencing is presented that uses a novel procedure for generating nested 'deletions' within inserts of single-stranded clones. In this method, single-stranded template, sequencing primer, and the Klenow fragment of Escherichia coli DNA polymerase I are used to initiate progressive DNA synthesis of the entire insert of the clone. By time-dependent sampling and pooling of intermediates from the synthesis reaction a series of nested double-stranded DNA subfragments of the insert can be created. Nested subclones are then produced by S1-endonuclease treatment and oriented subcloning methods. First, smaller quantities of template DNA can be used, equivalent to a fraction of a small DNA sequencing prep. Second, it works with single-stranded M13 phage DNA rather than requiring the preparation of double-stranded replicative form DNA as in ExoIII-based methods. Third, the 'deletions' it generates can span areas of simple nucleotide sequence or secondary structure that often halt digestion in the single-stranded exonuclease-based method. Last, the method is adaptable to a larger variety of insert cloning sites than the ExoIII-based method. The main disadvantage of the method is that, due to the lower efficiency of subcloning larger DNA fragments, subclone inserts larger than 3 kb are generated only infrequently.

Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease.

The virally encoded protease of human immunodeficiency virus is responsible for the processing of the gag and gag-pol polyprotein precursors to their mature polypeptides. Since correct processing of the viral polypeptides is essential for the production of infectious virus, HIV protease represents a potential target for therapeutic agents that may prove beneficial in the treatment of AIDS. In this study, full-length gag polyprotein has been synthesized in vitro to serve as a substrate for bacterially expressed HIV-1 protease. Expression of the protease in E. coli from the lac promoter was enhanced approximately five-fold by deletion of a potential hairpin loop upstream from the codon determining the amino terminus of mature protease. Extracts of induced cultures of E. coli harboring a protease-containing plasmid served as the source of protease activity. The gag polyprotein synthesized in vitro was cleaved by such lysates, producing fragments corresponding in size to p17 plus p24 and mature p24. Immunoprecipitations with monoclonal antibodies to p17 and p24 polypeptides suggest that initial cleavage of gag polyprotein occurs near the p24-p15 junction. The proteolysis was inhibited by pepstatin with an IC50 of 0.15 mM for cleavage at the p24-p15 junction and 0.02 mM for cleavage at the p17-p24 junction.

cis-Acting sequences in addition to donor and acceptor sites are required for template switching during synthesis of plus-strand DNA for duck hepatitis B virus.

A characteristic of all hepadnaviruses is the relaxed-circular conformation of the DNA genome within an infectious virion. Synthesis of the relaxed-circular genome by reverse transcription requires three template switches. These template switches, as for the template switches or strand transfers of other reverse-transcribing genetic elements, require repeated sequences (the donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. The mechanism for each of the template switches in hepadnaviruses is poorly understood. To determine whether sequences other than the donor and acceptor sites are involved in the template switches of duck hepatitis B virus (DHBV), a series of molecular clones which express viral genomes bearing deletion mutations were analyzed. We found that three regions of the DHBV genome, which are distinct from the donor and acceptor sites, are required for the synthesis of relaxed-circular DNA. One region, located near the 3' end of the minus-strand template, is required for the template switch that circularizes the genome. The other two regions, located in the middle of the genome and near DR2, appear to be required for plus-strand primer translocation. We speculate that these cis-acting sequences may play a role in the organization of the minus-strand DNA template within the capsid particle so that it supports efficient template switching during plus-strand DNA synthesis.