Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction.

We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.     

Genetic relationship between the Mus cervicolor M432 retrovirus and the Mus Musculus intracisternal type A particle

The genetic relationship between the retrovirus-like intracisternal type A particle (IAP) from Mus musculus and the novel retrovirus (M432) from M. cervicolor has been determined by heteroduplex and restriction endonuclease analyses of molecular clones of the respective genomes. We have found a major homology region (3.7 kilobase pairs) which probably begins near the 3' end of the M432 gag gene, spans the pol gene, and ends in the env gene. A second region (0.6 kilobase pairs) of weak homology was also observed adjacent to the 3' long terminal repeats of the respective genomes. The IAP genome is well conserved in the cellular DNA of all species of the genus Mus. In contrast, cellular DNA sequences related to the 5' end of the M432 genome, which shares no homology with the IAP genome, are found only in M. cervicolor and the closely related species M. cookii. These results suggest that the infectious M432 retroviral genome arose as a result of a recombinational event(s) between the IAP genome and another, as yet unidentified, class of retrovirus-related sequences or other cellular sequences.     

Isolation and characterization of IS elements repeated in the bacterial chromosome

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.     

Nucleotide sequence analysis of the long terminal repeat of avian myeloblastosis virus and adjacent host sequences.

The nucleotide sequence of the integrated avian myeloblastosis virus long terminal repeat has been determined. The sequence is 385 base pairs long and is present at both ends of the viral DNA. The cell-virus junctions at each end consist of a 6-base-pair direct repeat of cell DNA next to the inverted repeat of viral DNA. The long terminal repeat also contains promoter-like sequences, an mRNA capping site, and polyadenylation signals. Several features of this long terminal repeat suggest a structural and functional similarity with sequences of transposable and other genetic elements. Comparison of these sequences with long terminal repeats of other avian retroviruses indicates that there is a great variation in the 3' unique sequence (U3), whereas the 5' specific sequences (U5) and the R region are highly conserved.     

The 5' ends of Hantaan virus (Bunyaviridae) RNAs suggest a prime-and-realign mechanism for the initiation of RNA synthesis.

We examined the 5' ends of Hantaan virus (HTN) genomes and mRNAs to gain insight into the manner in which these chains were initiated. Like those of all members of the family Bunyaviridae described so far, the HTN mRNAs contained 5' terminal extensions that were heterogeneous in both length and sequence, presumably because HTN also "cap snatches" host mRNAs to initiate the viral mRNAs. Unexpectedly, however, almost all of the mRNAs contained a G residue at position -1, and a large fraction also lacked precisely one of the three UAG repeats at the termini. The genomes, on the other hand, commenced with a U residue at position +1, but only 5' monophosphates were found here, indicating that these chains may not have initiated with UTP at this position. Taken together, these unusual findings suggest a prime-and-realign mechanism of chain initiation in which mRNAs are initiated with a G-terminated host cell primer and genomes with GTP, not at the 3' end of the genome template but internally (opposite the template C at position +3), and after extension by one or a few nucleotides, the nascent chain realigns backwards by virtue of the terminal sequence repeats, before processive elongation takes place. For genome initiation, an endonuclease, perhaps that involved in cap snatching, is postulated to remove the 5' terminal extension of the genome, leaving the 5' pU at position +1.     

Recognition and cleavage of the bacteriophage P1 packaging site (pac). II. Functional limits of pac and location of pac cleavage termini

Bacteriophage P1 initiates the processive packaging of its DNA at a unique site called pac. We show that a functional pac site is contained within a 161 base-pair segment of P1 EcoRI fragment 20. It extends from a position 71 base-pairs to a position 232 base-pairs from the EcoRI-22 proximal side of that fragment. The 3' and 5' pac termini are located centrally within that 161 base-pair region and are distributed over about a turn of the DNA helix. The DNA sequence of the terminus region is shown below, with the large arrows indicating the positions of termini that are frequently represented in the PI population and the small arrows indicating the positions of termini that are rarely represented in the P1 population. (Sequence: in text). Digestion of P1 virus DNA with EcoRI generates two major EcoRI-pac fragments, which differ in size by about five or six base-pairs. While the structure and position of the double-stranded pac ends of these fragments have not been determined precisely, the 5' termini at those ends probably correspond to the two major pac cleavage sites in the upper strand of the sequences shown above. The 161 base-pair pac site contains the hexanucleotide sequence 5'-TGATCAG-3' repeated four times at one end and three times at the other. Removal of just one of those elements from either the right or left ends of pac reduces pac cleavage by about tenfold. Moreover, the elements appear to be additive in their effect on pac cleavage, as removal of one and a half elements or all three elements from the right side of pac reduces pac cleavage 100-fold, and greater than 1000-fold, respectively.     

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.     

The relationships between the 5'' end repeats and the largest members of the L1 interspersed repeated family in the mouse genome.

Analysis of a few large L1 elements has revealed two types of tandem repeats at the 5' end: A and F. In this study, the relationships between these repeats and a series of large L1 elements has been analysed. Most of cloned L1 repeats were shown to lack either A or F sequences at their 5' ends. F sequences are found less frequently associated than A sequences to the 5' ends of L1 and an evolutionary comparison shows that the A type was introduced more recently during the evolution of the mouse genome than the F type.     

Recognition and elongation of telomeres by telomerase

Telomeres stabilize chromosomal ends and allow their complete replication in vivo. In diverse eukaryotes, the essential telomeric DNA sequence consists of variable numbers of tandem repeats of simple, G + C rich sequences, with a strong strand bias of G residues on the strand oriented 5' to 3' toward the chromosomal terminus. This strand forms a protruding 3' over-hang at the chromosomal terminus in three different eukaryotes analyzed. Analysis of yeast and protozoan telomeres showed that telomeres are dynamic structures in vivo, being acted on by shortening and lengthening activities. We previously identified and partially purified an enzymatic activity, telomere terminal transferase, or telomerase, from the ciliate Tetrahymena. Telomerase is a ribonucleoprotein enzyme with essential RNA and protein components. This activity adds repeats of the Tetrahymena telomeric sequence, TTGGGG, onto the 3' end of a single-stranded DNA primer consisting of a few repeats of the G-rich strand of known telomeric, and telomere-like, sequences. The shortest oligonucleotide active as a primer was the decamer G4T2G4. Structural analysis of synthetic DNA oligonucleotides that are active as primers showed that they all formed discrete intramolecular foldback structures at temperatures below 40 degrees C. Addition of TTGGGG repeats occurs one nucleotide at a time by de novo synthesis, which is not templated by the DNA primer. Up to 8000 nucleotides of G4T2 repeats were added to the primer in vitro. We discuss the implications of this finding for regulation of telomerase in vivo and a model for telomere elongation by telomerase.     

Sequence and hairpin structure of an inverted repeat series at termini of the Physarum extrachromosomal rDNA molecule

The termini of the 61 kb palindromic rDNA molecules of Physarum polycephalum possess a series of multiple inverted repeats in which are located specific single-strand gaps and tightly attached protein. After treating rDNA with S1 nuclease, we have cloned several 5 kb Eco RI terminal restriction fragments. Sequencing of more than 800 nucleotides from the end of one such clone reveals the presence of six to ten tandemly repeated units averaging 140 +/- 4 bp in length and flanked by Hae III sites. Each 140 nucleotide repeat unit can form thermodynamically stable hairpin structures based on complex internal palindromic components. When the specific gap sequence CCCTA is present, it is located near the apex of a hairpin component. These secondary structures are formed in growing plasmodia, as seen in electron micrographs of native rDNA molecules, which also reveal apparent recombination forms involving rDNA ends and noncontiguous DNA segments. Recombination initiated at terminal single-strand hairpin loops can result in genetic exchange of ribosomal gene sequences and can lead to completion of 5' nucleotide sequences at ends of newly replicated rDNA molecules.     

The gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) from the chicken was isolated from a recombinant library containing the chicken genome in phage lambda Charon 4A. The isolated clone, lambda PCK1cc, contains the complete gene for the enzyme as well as both 5' and 3' flanking sequences. The gene is approximately 8 kilobases in length divided into 8 exons, as demonstrated by restriction endonuclease mapping and DNA-RNA heteroduplex analysis. Southern blotting of chicken chromosomal DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1cc. The phosphoenolpyruvate carboxykinase gene is present as a single copy in the haploid chicken genome. The 5' region of the gene was defined by S1 nuclease mapping and by sequencing. Two mRNA species with discrete 5' ends were observed using S1 nuclease mapping. The ratio between the amounts of these multiple forms of mRNA is the same in chicken kidney and liver and is not affected by induction of the enzyme mRNA by cAMP. Examination of sequence homologies with the gene for rat cytosolic phosphoenolpyruvate carboxykinase indicates a putative control region contained in flanking sequences at the 5' end of the gene.     

Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus.

BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.     

Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae.

Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer). We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins. The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides. A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN. When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends. The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used. Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro. We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer. The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing.     

Complete nucleotide sequence of the rabbit beta-like globin gene cluster. Analysis of intergenic sequences and comparison with the human beta-like globin gene cluster

The nucleotide sequence of the entire beta-like globin gene cluster of rabbits has been determined. This sequence of a continuous stretch of 44.5 x 10(3) base-pairs (bp) starts about 6 x 10(3) bp upstream from epsilon (the 5'-most gene) and ends about 12 x 10(3) bp downstream from beta (the 3'-most gene). Analysis of the sequence reveals that: (1) the sequence is relatively A + T rich (about 60%); (2) regions with high G + C content are associated with OcC repeats, a short interspersed repeated DNA in rabbits; (3) the distribution of polypurines, polypyrimidines and alternating purine/pyrimidine tracts is not random within the cluster; (4) most open reading frames are associated with known globin coding regions, OcC repeats or long interspersed repeats (L1 repeats); (5) the most prominent open reading frames are found in the L1 repeats; (6) different strand asymmetries in base composition are associated with embyronic and adult genes as well as the tandem L1 repeats at the 3' end of the cluster; and (7) essentially all the repeats appear to have been inserted by a transposon mechanism. A comparison of the sequence with itself by a dot-plot analysis has revealed nine new members of the OcC family of repeats in addition to the six previously reported. The OcC repeats tend to be clustered, particularly in the epsilon-gamma and gamma-psi delta intergenic regions. Dot-plot comparisons between the rabbit and the human clusters have revealed extensive sequence matches. Homology starts about 6 x 10(3) bp 5' to epsilon or as far upstream as the rabbit sequence is available. It continues throughout the entire cluster and stops about 0.7 x 10(3) bp 3' to beta, at which point several repeats have inserted in both rabbits and humans. Throughout the gene cluster, the homology is interrupted mainly by insertions or deletions in either the rabbit or the human genome. Almost all of the insertions are of known short or long repeated DNAs. The positions of the insertions are different in the two gene clusters, which indicates that both short and long repeats have been transposing throughout the genome for the time since the mammalian radiation. An alignment of rabbit and human sequences allows the calculation of the substitution rate around epsilon. Sequences far removed from the gene are evolving at a rate equivalent to the pseudogene rate, although some short regions show an apparently higher rate.(ABSTRACT TRUNCATED AT 400 WORDS)