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A V Furano

Publications and source records attributed to A V Furano.

At least 37 records · Page 2Linked to original sources
PubMed1988

The structure of the regulatory region of the rat L1 (L1Rn, long interspersed repeated) DNA family of transposable elements.

Here we report the DNA structure of the left 1.5 kb of two newly isolated full length members of the rat L1 DNA family (L1Rn, long interspersed repeated DNA). In contrast to earlier isolated rat L1 members, both of these contain promoter-like regions that are most likely full length. In addition, the promoter-like region of both members has undergone a partial tandem duplication. A second internal region of the left end of one of the reported members is also tandemly duplicated. The propensity of the left end of rat L1 elements to undergo this form of genetic rearrangement, as well as other structural features revealed by the present work, is discussed in light of the fact that during evolution the otherwise conserved mammalian L1 DNA families have each acquired completely different promoter-like regions. In an accompanying paper [Nur, I., Pascale, E., and Furano, A. V. (1988) Nucleic Acids Res. 16, submitted], we report that one of the rat promoter-like regions can function as a promoter in rat cells when fused to the Escherichia coli chloramphenicol acyltransferase gene.

Amino Acid Sequence↗
PubMed1988

The left end of rat L1 (L1Rn, long interspersed repeated) DNA which is a CpG island can function as a promoter.

Here we report that the 600 bp promoter-like region at the left end of a newly isolated and characterized rat L1 DNA element can activate the prokaryotic chloramphenicol acyltransferase gene in a rat cell line. Activation only occurs when the promoter region is oriented to the transferase gene as it is to the L1 protein encoding sequences and is 75% inhibited by methylation of just 5 of the 22 CpGs present in the promoter. The G + C rich promoter contains enough CpGs to qualify it as a CpG island, but in contrast to other CpG islands, genomic L1 promoters are fully methylated in both somatic cell and sperm DNA as judged by restriction enzyme analysis. Partial demethylation of the genomic promoters by treatment with 5-azacytidine failed to produce discrete L1 transcripts. The relationship of methylation to the evolutionary history and fate of the rat L1 promoter is discussed.

Animals↗
PubMed1988

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed.

Animals↗
PubMed1987

DNA synthesis arrest sites at the right terminus of rat long interspersed repeated (LINE or L1Rn) DNA family members.

An approximately equal to 150-bp GC-rich (approximately equal to 60%) region is at the right end of rat long interspersed repeated DNA (LINE or L1Rn) family members. We report here that one of the DNA strands from this region contains several non-palindromic sites that strongly arrest DNA synthesis in vitro by the prokaryotic Klenow and T4 DNA polymerases, the eukaryotic alpha polymerase, and AMV reverse transcriptase. The strongest arrest sites are G-rich (approximately equal to 70%) homopurine stretches of 18 or more residues. Shorter homopurine stretches (12 residues or fewer) did not arrest DNA synthesis even if the stretch contains 11/12 G residues. Arrest of the prokaryotic polymerases was not affected by their respective single strand binding proteins or polymerase accessory proteins. The region of duplex DNA which contains DNA synthesis arrest sites reacts with bromoacetaldehyde when present in negatively supercoiled molecules. By contrast, homopurine stretches that do not arrest DNA synthesis do not react with bromoacetaldehyde. The presence of bromoacetaldehyde-reactive bases in a G-rich homopurine-containing duplex under torsional stress is thought to be caused by base stacking in the homopurine strand. Therefore, we suggest that base-stacked regions of the template arrest DNA synthesis.

Animals↗
PubMed1986

Target sites for the transposition of rat long interspersed repeated DNA elements (LINEs) are not random.

The long interspersed repeated DNA family of rats (LINE or L1Rn family) contains about 40,000 6.7-kilobase (kb) long members (1). LINE members may be currently mobile since their presence or absence causes allelic variation at three single copy loci (2, 3): insulin 1, Moloney leukemia virus integration 2 (Mlvi-2) (4), and immunoglobulin heavy chain (Igh). To characterize target sites for LINE insertion, we compared the DNA sequences of the unoccupied Mlvi-2 target site, its LINE-containing allele, and several other LINE-containing sites. Although not homologous overall, the target sites share three characteristics: First, depending on the site, they are from 68% to 86% (A+T) compared to 58% (A+T) for total rat DNA (5). Depending on the site, a 7- to 15-bp target site sequence becomes duplicated and flanks the inserted LINE member. The second is a version (0 or 1 mismatch) of the hexanucleotide, TACTCA, which is also present in the LINE member, in a highly conserved region located just before the A-rich right end of the LINE member. The third is a stretch of alternating purine/pyrimidine (PQ). The A-rich right ends of different LINE members vary in length and composition, and the sequence of a particularly long one suggests that it contains the A-rich target site from a previous transposition.

Animals↗
PubMed1986

Structure of the highly repeated, long interspersed DNA family (LINE or L1Rn) of the rat.

We present the DNA sequence of a 6.7-kilobase member of the rat long interspersed repeated DNA family (LINE or L1Rn). This member (LINE 3) is flanked by a perfect 14-base-pair (bp) direct repeat and is a full-length, or close-to-full-length, member of this family. LINE 3 contains an approximately 100-bp A-rich right end, a number of long (greater than 400-bp) open reading frames, and a ca. 200-bp G + C-rich (ca. 60%) cluster near each terminus. Comparison of the LINE 3 sequence with the sequence of about one-half of another member, which we also present, as well as restriction enzyme analysis of the genomic copies of this family, indicates that in length and overall structure LINE 3 is quite typical of the 40,000 or so other genomic members of this family which would account for as much as 10% of the rat genome. Therefore, the rat LINE family is relatively homogeneous, which contrasts with the heterogeneous LINE families in primates and mice. Transcripts corresponding to the entire LINE sequence are abundant in the nuclear RNA of rat liver. The characteristics of the rat LINE family are discussed with respect to the possible function and evolution of this family of DNA sequences.

Animals↗
PubMed1985

Insertion of long interspersed repeated elements at the Igh (immunoglobulin heavy chain) and Mlvi-2 (Moloney leukemia virus integration 2) loci of rats.

Restriction enzyme analysis of normal DNA derived from individual rats of the National Institutes of Health outbred Osborn-Mendel colony revealed that two independent single-copy loci, the Igh (immunoglobulin heavy chain) locus and the Mlvi-2 (Moloney leukemia virus integration 2) locus, a putative oncogene, are polymorphic (i.e., exhibit allelic variation). The polymorphism at both loci was due to the presence or absence of a long interspersed repeated DNA element (LINE). The LINE insertion in the Igh locus occurred in the joining (J) region, which is involved in the physiological rearrangement of this locus. The LINE insertion in the Mlvi-2 locus has occurred approximately 6 kilobases from the region of provirus integration in Moloney murine leukemia virus-induced rat thymomas. The two inserts are colinear with each other and with other randomly selected cloned copies of the rat LINE family, the general characteristics of which we also present. LINE insertion in the Mlvi-2 locus was observed in several rat strains, established from independent rat colonies, suggesting that LINE-containing Mlvi-2 alleles may be widespread in the rat population. LINE insertion in the Igh locus was observed in 1 of 27 rats. The detection of a LINE-related polymorphism at two nonselected loci indicates that LINEs are transposable. The presence or absence of these long (greater than 5 kilobases), highly transcribed elements at single-copy loci could have profound effects on gene activity. Furthermore, LINE-containing single-copy loci could be affected by homologous interaction between the resident LINE and any of the other 50,000 or so copies of these elements in the rat genome.

Animals↗
PubMed1985

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

Alleles↗
PubMed1984

Highly repeated DNA families in the rat.

We have analyzed the repeated DNA fraction of the rat by characterizing approximately 500 repeat DNA-containing clones using hybridization to a variety of rodent nucleic acids. To facilitate this analysis we devised a method whereby the cloned DNA is transferred to nitrocellulose paper by blotting directly out of colonies of the bacterial clones. In addition to identifying repeated sequences of potential interest (e.g. those transcribed in a tissue-specific manner, or those that are highly conserved in non-rat genomes), we found that, in contrast to what is revealed by the reassociation of rat DNA (e.g. Pearson, W. R., Wu, J. R., and Bonner, J. (1978) Biochemistry 17, 51-59), the rat genome contains a number of different highly repeated (greater than 50,000 copies) sequences. We distinguished the different highly repeated sequences both by their hybridization to different nucleic acids as well as by DNA sequence determination. The highly repeated sequences shared three characteristics that distinguished each of them from the 100,000-member rat satellite I family: (i) they were recovered less often in the cloned repeat DNA library than expected from their copy number in the rat genome; (ii) they reannealed abnormally slowly for their copy number even though they are not significantly divergent; and (iii) they are transcribed in one or more rat tissues. The implications of these findings for the organization of repeated sequences in the rat genome are discussed.

Adrenal Gland Neoplasms↗
PubMed1984

The spread of sequence variants in Rattus satellite DNAs.

The genus Rattus has two related families of satellite DNA: Satellite I consists of tandem arrays of a 370 base pair repeat unit which is a dimer of two 185 base pair portions (a, b) which are about 60% homologous. Satellite I' consists of tandem arrays of a 185 base pair repeat unit (a') which is about 85% homologous to a and 60% homologous to b. R. norvegicus contains only satellite I but R. rattus contains both satellites I and I'. We examined certain aspects of satellite DNA evolution by comparing the spacing at which variant repeat units of each satellite have spread among non-variant repeat units in these two species. With but one exception, in R. rattus, 15 different variant repeat units have spread among non-variant repeat units of satellite I, with a spacing equal to the length of the (a,b) dimer. Similarly, fourteen different variant repeat units of the monomeric satellite I' have mixed among non-variant repeat units with a spacing equal to the length of the (a') monomer. These results suggest that a mechanism involving homologous interaction among satellite sequences could account for the spread of variant family members. We also found that a sequence variant present in certain portions of the dimeric repeat unit of satellite I is more efficiently amplified (or less efficiently corrected) than variants occurring in other regions. This was not true for the monomeric repeat unit of satellite I'.

Animals↗
PubMed1984

A convenient method for locating sets of related short sequences in DNA sequences of any length.

In investigating sequence variants in a family of highly repeated rat DNA, we needed to search the consensus sequence of the repeat unit of this family for short sequences which would become, with one base change, recognition sites for various restriction endonucleases. To do this, we have designed a pair of programs to search DNA sequences of any length for sets of related short sequences, allowing user-specified mismatches in the short sequence. Since putative regulatory regions are generally short sequences, these programs are also useful for locating all possible versions of such sequences in any given DNA. We describe the programs, and present results of searches using the programs.

Base Sequence↗
PubMed1983

The independent evolution of two closely related satellite DNA elements in rats (Rattus).

We have identified and determined the sequence and organization of a new rat satellite DNA in Rattus rattus, the roof rat. This satellite DNA, which we call R. rattus satellite I', consists of tandem arrays of a 185 base pair (bp) repeat unit that we call a'. a' is 86% homologous to a 185 bp portion of the 370 bp repeat unit of the previously described rat satellite [Pech et al. (1979) Nucleic Acids Res. 7, 417-432] present in the common laboratory rat species, R. norvegicus. We can thereby distinguish two 185 bp portions of the satellite I 370 bp repeat unit: "a" (homologous to a') and "b". Satellite I has the structure (a,b)n, and satellite I' has the structure (a')n. Like a, a' is only about 60% homologous to b and fails to hybridize to b. Although R. norvegicus and R. rattus contain about the same total concentration of satellite sequences, R. norvegicus contains essentially only the a,b type (satellite I), whereas R. rattus contains a' type (satellite I') and lesser amounts of the a,b type (satellite I). The a,b type (satellite I) in R. rattus is very similar to that in R. norvegicus as judged both by hybridization and by the presence of all but one of the major restriction enzyme sites that characterize the R. norvegicus satellite I. In R. rattus the a' and a,b repeat units are not detectably present in the same tandem array. All of the sequence differences between a' (R. rattus) and a (R. norvegicus) can be accounted for by simple base substitutions, and the implication of this and other features of rat satellite DNA structure for satellite DNA evolution are discussed.

Animals↗
PubMed1981

Regulation of the synthesis of E. coli elongation factor Tu.

Rapidly growing E. coli with two active genes (tufA, tufB) for elongation factor (EF) Tu contains three times as much EF-Tu (tuf) mRNA as EF-G (fus) mRNA on a molar basis, but about seven times as much EF-Tu as EF-G or ribosomes. The concentration and translational efficiency of fus (EF-G) mRNA is about that for ribosomal protein mRNAs. The high molar concentration of EF-Tu relative to EF-G or ribosomes is achieved in part by translating tuf mRNA more efficiently than these other mRNAs. In a tufA+ tufB-: : Mu strain, the tuf:fus mRNA ratio is 1, but the concentration of EF-Tu and tuf mRNA is the same as in the wild-type strain. Thus cells with only an active tufA gene increase the concentration but not the translational efficiency of tuf mRNA. In such cells the concentration of fus mRNA is almost three times that in the wild-type strain. Because the tufA gene is distal to but cotranscribed with the fus gene as part of the four gene str operon, the wild-type concentration of tuf mRNA in these tufB- cells must be produced by increasing the concentration of transcript corresponding to the entire str operon. Thus transcription of the tufA gene can only proceed from the str promoter. Extracts of the tufB- cells contain tuf transcripts that correspond not only in size to the entire 4.5 kb str operon, but also to the size (approximately 1 kb) of a tuf gene. Our evidence suggests that this 1 kb tuf transcript is derived by posttranscriptional modification of the primary str operon transcript and that this modification could in part explain the high translational efficiency of tuf mRNA.

Bacterial Proteins↗
PubMed1981

The conservation of DNA sequences over very long periods of evolutionary time. Evidence against intergeneric chromosomal transfer as an explanation for the presence of Escherichia coli tuf gene sequences in taxonomically-unrelated prokaryotes.

In the present study we tried to determine whether the presence of DNA sequences homologous to the Escherichia coli tuf gene (encodes peptide chain elongation factor Tu) in many taxonomically-unrelated prokaryotes is due to selective pressure for these sequences or due to the transfer of chromosomal material subsequent to the divergence of the genera from their progenitors. We found that the degree of sequence homology to the DNA immediately adjacent to the E. coli tuf A gene is either nonexistent or much less than that found for the tuf gene. Furthermore, the tuf-homologous sequences present in one prokaryote were found to be in large part the same as or a subset of those present in others. That is, various prokaryotes share a common subset of tuf-homologous sequences. These findings suggest that strong selective pressure and not recent intergeneric chromosomal transfer is responsible for the ubiquitous presence of certain tuf-homologous sequences. Because the genetic code is degenerate, DNA sequence need not be conserved to conserve protein sequence. Therefore, if the only function of these sequences is to encode protein, their persistence must mean that in some instances codon sequence is selected for.

Bacteria↗
PubMed1981

Duplication of the tuf gene, which encodes peptide chain elongation factor Tu, is widespread in Gram-negative bacteria.

The tuf gene which encodes peptide chain elongation factor Tu was found to be duplicated in nine enteric and four nonenteric gram-negative bacteria, but present only in one copy in two gram-positive genera. In two of the nonenteric gram-negative genera, Pseudomonas and Caulobacter, the duplicate tuf genes were found to be very close together on the chromosome, which contrasts with the situation in Escherichia coli, where they are more than 660 kilobases apart.

Chromosome Mapping↗
PubMed1978

The peptide chain elongation factor genes tufA and fus of Escherichia coli are intimately related physically.

Recent work from several laboratories has established the following points about the synthesis of the polypeptide chain elongation factors Tu and G in Escherichia coli. (i) Elongation factor Tu is the product of duplicate, highly conserved genes, tufA and tufB, which are widely separate parts of the chromosome. (ii) The molar concentration of this factor is considerably higher than that of elongation factor G which is encoded by the fus gene. (iii) Although the tufA and fus genes are close together and can be co-transcribed in the direction from fus to tufA, the tufA gene product is synthesized at several times the rate of the fus gene product. In an attempt to understand what mechanism(s) could account for the differential expression of the tufA and fus genes, we sought to obtain more precise information on the physical relationship of these genes. By examining heteroduplexes between restriction endonuclease-generated fragments of DNA containing the tufA, fus, and tufB genes, we have demonstrated that the fus and tufA genes are intimately related physically in one of two possible arrangements. Either the NH2-terminal region of the tufA gene is contiguous with the COOH-terminal region of the fus gene or the beginning of the tufA gene overlaps part of the fus gene. These results mean that if the tufA gene is always co-transcribed with the fus gene, then some mechanism must allow the tufA portion of the transcript to be translated more often than the fus gene portion of the transcript.

DNA, Bacterial↗