Biomedical subjects

S Subramani

Publications and source records attributed to S Subramani.

At least 109 records · Page 6Linked to original sources

PubMed1989

Human papovavirus BK early gene regulation in nonpermissive cells.

The human papovavirus BK latently infects a majority of the population worldwide, and its DNA has been found in human tumor tissue. BKV is known to be highly oncogenic in rodents, and is capable of transforming cells in vitro. Rearrangements in the transcriptional regulatory sequences controlling expression of the transforming early gene, T antigen, are known to affect both the tumorigenic and transforming properties of this virus. Little is known about the mechanism by which this occurs. We have examined several aspects of BKV early promoter/enhancer regulation in cell types which the virus transforms, baby hamster kidney (BHK) and newborn rat kidney (NRK) cells, and compare them to the same processes in monkey kidney CV1 cells. We find that BKV early transcriptional efficiency requires the same enhancer repeat elements in all three cell types, but that requirements for sequences to the early and late side of these repeats vary between these cells. While the BKV T antigen was found to repress early gene expression from the BKV early promoter in CV1 cells, this effect was lower in BHK cells and essentially absent in NRK cells. The impaired autoregulation observed in rodent cells may be the result of inefficient T antigen production in these cells. DNA replication from the BKV origin was not detected in either BHK or NRK cells. Finally, we find no correlation between the efficiency of the BKV early regulatory region in directing gene expression and the ability to transform NRK cells.

Animals↗

PubMed1989

A naturally occurring deletion in the enhancer repeats of the human papovavirus BK optimizes early enhancer function at the expense of late promoter activity.

The late promoter of the human papovavirus BK (prototype) is contained within the same region of DNA as the early promoter enhancer. This region, consisting of a 68-50-68-bp repeat and the flanking region to the late side, contains elements important for both late promoter activity and enhancer function for the early transcription unit. We have studied the importance of the naturally occurring 18-bp deletion in the middle repeat by constructing three repeats containing every possible combination of the 50- and 68-bp segments and testing their abilities to promote late transcription and activate an enhancerless SV40 early promoter. We find that the wild-type configuration of repeats gives optimal enhancer function, but is less active than other combinations for late transcription. This suggests that different interactions between the transcription elements contained within the same region of DNA are responsible for modulation of early and late transcription.

BK Virus↗

PubMed1989

Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species.

The DNA polymerase chain reaction was developed for in vitro amplification of specific DNA sequences, and it has been used for a wide variety of purposes in several fields. We have developed an application of the polymerase chain reaction that is useful for the isolation of partial cDNA or genomic clones of conserved genes. We used this technique to clone the gene encoding the iron protein subunit (27 kDa) of succinate dehydrogenase (EC 1.3.5.1) from several species, including human, rat, Drosophila melanogaster, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Mixed oligonucleotide primers corresponding to two conserved regions of the protein were used in conjunction with genomic and cDNA templates in the reaction. The primers contained all possible nucleotide combinations that could encode the corresponding peptide sequences. These oligonucleotide mixtures contained 262,144 (2(18] and 8192 (2(13] unique sequences, respectively. Use of the polymerase chain reaction for homology probing allows one to utilize more complex mixtures of oligonucleotides as probes than is possible with filter hybridization screening techniques. In addition, the polymerase chain reaction offers the advantage of synthesizing the DNA product directly, in some cases obviating the need to construct cDNA or genomic libraries. This application of the polymerase chain reaction should be useful not only for the identification of conserved genes in a variety of species but also for the isolation of previously unknown members of gene families.

Amino Acid Sequence↗

PubMed1989

A conserved tripeptide sorts proteins to peroxisomes.

The firefly luciferase protein contains a peroxisomal targeting signal at its extreme COOH terminus (Gould et al., 1987). Site-directed mutagenesis of the luciferase gene reveals that this peroxisomal targeting signal consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine. When this tripeptide is appended to the COOH terminus of a cytosolic protein (chloramphenicol acetyltransferase), it is sufficient to direct the fusion protein into peroxisomes. Additional mutagenesis experiments reveal that only a limited number of conservative changes can be made in this tripeptide targeting signal without abolishing its activity. These results indicate that peroxisomal protein import, unlike other types of transmembrane translocation, is dependent upon a conserved amino acid sequence.

Amino Acid Sequence↗

PubMed1989

Analysis of origin of DNA replication of human papovavirus BK.

We have analyzed the cis-acting sequence elements and properties of the origin of DNA replication of human papovavirus BK (BKV). The precise boundaries of the origin varied, depending on the cell type and the viral T antigen used for assay. The BKV minimal origin of replication consisted of an inverted repeat, T-antigen-binding site II, and a 20-base-pair AT block when assayed in monkey kidney CV1 and HeLa cells by using the BKV T antigen. This 76-base-pair minimal origin did not replicate in COS cells in the presence of the simian virus 40 (SV40) T antigen. Unlike that from the SV40 minimal origin, replication from the BKV minimal origin was not enhanced by BKV ori-flanking sequences in CV1 or HeLa cells, using the BKV T antigen. BKV ori-flanking sequences did activate the SV40 minimal origin of replication in COS cells and relieved the orientation-dependent property of this origin. Finally, the BKV T antigen was found to autoregulate activity of the BKV early transcriptional regulatory region. The BKV origin of replication shows similarities to and differences from those of the related viruses SV40 and polyomavirus, suggesting that the proteins involved in the initiation of replication interact with origin sequences differently in these viruses.

Animals↗

PubMed1988

Do transcriptional enhancers also augment DNA replication?

Enhancers are DNA elements that augment transcription in cis, independent of distance and orientation. Evidence such as hormone dependent neoplastic cell growth and the stimulation of viral replication by sequences present in enhancers suggests that enhancers may also directly affect DNA replication. We tested this hypothesis in recombinant plasmids by asking whether sequences that stimulated DNA replication shared the properties of transcriptional enhancers. The homologous simian virus 40 (SV40) core enhancer was ligated either adjacent to or 2.6 kb distant from the SV40 minimal origin of replication (ori) in both orientations. Plasmids were transfected into T antigen producing COS cells, and episomal DNA was harvested for quantitation of replication. Replication could be assessed either as accumulation of fmol of MboI sensitive progeny DNA, or as a transition in % DNA in replicated (MboI sensitive) versus unreplicated (DpnI sensitive) form. The two measures were related exponentially (r = 0.86). The SV40 enhancer augmented replication 1.5-10 fold. The effect was time dependent, distance dependent (only the adjacent enhancer locus stimulated replication), partially orientation dependent, and enhancer copy number independent. Phorbol ester did not affect replication. The heterologous glucocorticoid enhancer had no effect on replication. We conclude that the SV40 enhancer's cis-effect on replication seems to be dependent on the close proximity to the replication origin of specific homologous sequences within the enhancer, rather than a typical enhancer-like effect.

Animals↗

PubMed1988

Firefly luciferase as a tool in molecular and cell biology.

The unique properties of firefly luciferase and the cloning of the gene for this enzyme have spawned a number of novel applications of this protein. We summarize a few of these applications including its use as a reporter gene, as a model for the study of protein import into peroxisomes, and as a component of a heterologous gene expression system.

Animals↗

PubMed1988

The late promoter of the human papovavirus BK is contained within the early promoter enhancer region.

We have analyzed the sequences necessary for late promoter function and mapped the late mRNA start sites of the human papovavirus BK (prototype). Our results show that, under both replicating and nonreplicating conditions, the BKV late promoter is contained within the same region defined as the enhancer for the early promoter. This region consists of three 68-bp repeats (the middle one of which has an 18-bp deletion) and a 66-bp region containing an enhancer element denoted as c, located to the late side of the 68-bp repeats. Deletions within the early enhancer domain indicate that elements of the late promoter are found throughout the entire region.

Antigens, Viral, Tumor↗

PubMed1988

Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins.

As part of an effort to understand how proteins are imported into the peroxisome, we have sought to identify the peroxisomal targeting signals in four unrelated peroxisomal proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl-CoA oxidase. Using gene fusion experiments, we have identified a region of each protein that can direct heterologous proteins to peroxisomes. In each case, the peroxisomal targeting signal is contained at or near the carboxy terminus of the protein. For catalase, the peroxisomal targeting signal is located within the COOH-terminal 27 amino acids of the protein. For hydratase:dehydrogenase, D-amino acid oxidase, and acyl-CoA oxidase, the targeting signals are located within the carboxy-terminal 15, 14, and 15 amino acids, respectively. A tripeptide of the sequence Ser-Lys/His-Leu is present in each of these targeting signals as well as in the peroxisomal targeting signal identified in firefly luciferase (Gould, S.J., G.-A. Keller, and S. Subramani. 1987. J. Cell Biol. 105:2923-2931). When the peroxisomal targeting signal of the hydratase:dehydrogenase is mutated so that the Ser-Lys-Leu tripeptide is converted to Ser-Asn-Leu, it can no longer direct proteins to peroxisomes. We suggest that this tripeptide is an essential element of at least one class of peroxisomal targeting signals.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1988

Linker scan analysis of the early regulatory region of human papovavirus BK.

BK virus (BKV) is a human papovavirus which latently infects a majority of the world population. Reactivation of this virus is associated with acute hemorrhagic cystitis, and BKV DNA has been found in human tumor tissue. BKV is one of many highly homologous papovaviruses, including simian virus 40 and JC virus, which display distinct host and cell-type specificities, transformation potentials, and pathologies. These differences are thought to be determined, in part, by the noncoding regulatory region of each virus, which contains the origin of replication and regulatory elements for both early and late gene expression. We have used linker scan mutants to map functional elements of a truncated BKV early promoter and enhancer and have studied the stereospecific requirements of these elements. We have also identified protein-binding regions through DNase protection studies. Our results show that a minimum of four elements are necessary for efficient early transcription, at least three of which correspond to DNase-protected domains. These protein-binding elements map to the TATA box and two nuclear factor 1 consensus sequences, one located within the enhancer repeat unit and the other located to the late side of the enhancer. The sequence of the fourth element is similar to the transcription factor Sp1 consensus sequence. Additional DNase-protected sites are centered over AP-1 and Sp1 consensus sequences. Finally, we find that the functional elements of the BKV early promoter and enhancer lack strict stereospecific requirements for efficient transcription.

BK Virus↗

PubMed1987

Correction of deletions in mammalian cells by gene conversion.

We have constructed substrates to study the conversion of deletions in mammalian cells both extrachromosomally and after the stable integration of the substrates into the chromosome. These substrates were designed to study gene conversion without the complication of reciprocal recombination events. The substrates contain insertion or deletion mutations of the neomycin resistance gene (neo) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to the mutated neo gene restores a functional neo gene. We have shown that extrachromosomally insertions of 10 bp or deletions of 22 or 167 bp are converted to wild-type at similar frequencies (1-6 X 10(-4)). Chromosomal gene conversion occurred at frequencies of about 10(-6)-10(-7) per cell generation. As expected from the experimental design, all recombination events analyzed in mammalian cells using these substrates appear to be due to gene conversion.

Animals↗

PubMed1987

Analysis of the early regulatory region of the human papovavirus BK.

BKV is a human papovavirus which latently infects a majority of the world population and whose DNA has been found in human tumor tissue. Along with simian virus 40 (SV40) and JCV, it is one of several highly homologous polyomaviruses which display distinct host ranges, tissue tropisms, and transformation potentials. Determination of these properties is thought to reside, in part, in the noncoding regulatory region of these viruses. We have studied the regulation of gene expression by the early promoter and enhancer of BKV. Our results show that the early promoter of BKV consists of elements found both to the early side of and within the proximal 18 bp of the first enhancer element itself. At least one BKV regulatory element appears to be downstream of the mRNA start sites. The BKV enhancer consists of two different types of elements, three direct repeats, and an element (denoted "c") found in the 30 bp to the late side of the distal repeat. When used with the BKV promoter the enhancer repeat elements were found to be redundant, optimal promoter activity requiring only two of the three repeats plus the c element. Using the heterologous SV40 promoter the optimal BKV enhancer consisted of either three repeats or two repeats plus the c element. Subfragments of the enhancer region were capable of partial activation of homologous and heterologous promoters. We conclude that the regulation of BKV early gene expression involves novel elements arranged in ways not previously described in other papovaviruses.

Acetyltransferases↗

PubMed1987

Firefly luciferase is targeted to peroxisomes in mammalian cells.

Although several enzymes known to reside in peroxisomes have been studied extensively, no cis-acting amino acid sequences involved in the transport of these proteins to peroxisomes have been described. As a first step towards the determination of a putative peroxisomal targeting sequence, we have expressed the cDNA encoding the firefly luciferase [Photinus-luciferin:oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] in monkey kidney cells and found that the product of the gene is transported to peroxisomes. Luciferase is derived from the firefly (Photinus pyralis) and is synthesized and stored in the cells of the firefly's lantern organ, where it is also found in peroxisomes. The fact that this protein is similarly targeted in cells from such different organisms suggests that the process of protein transport to peroxisomes has been highly conserved through evolution.

Animals↗

PubMed1987

Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase.

Translocation of proteins across membranes of the endoplasmic reticulum, mitochondrion, and chloroplast has been shown to be mediated by targeting signals present in the transported proteins. To test whether the transport of proteins into peroxisomes is also mediated by a peptide targeting signal, we have studied the firefly luciferase gene that encodes a protein transported to peroxisomes in both insect and mammalian cells. We have identified two regions of luciferase which are necessary for transport of this protein into peroxisomes. We demonstrate that one of these, region II, represents a peroxisomal targeting signal because it is both necessary and sufficient for directing cytosolic proteins to peroxisomes. The signal is no more than twelve amino acids long and is located at the extreme carboxy-terminus of luciferase. The location of the targeting signal for translocation across the peroxisomal membrane therefore differs from the predominantly amino-terminal location of signals responsible for transport across the membranes of the endoplasmic reticulum, chloroplast, or mitochondrion.

Acetyltransferases↗

PubMed1987

Firefly luciferase gene: structure and expression in mammalian cells.

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

Animals↗

PubMed1986

Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination.

Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian virus 40 from the chromosome of monkey COS cells to two different, extrachromosomally replicating, simian virus 40 DNA molecules lacking this 1,018-bp sequence. The ratio of rescued to unrecombined virus was as high as 10(-3). The rescued molecules, detected optimally 5 to 9 days after transfection of COS cells, had completely recovered the 1,018-bp DNA segment from the chromosome. The recombination event is proposed to occur either by double reciprocal recombination or by gene conversion between the chromosomal T-antigen gene and the extrachromosomal molecules missing the 1,018-bp sequence.

Animals↗

PubMed1986

Extrachromosomal and chromosomal gene conversion in mammalian cells.

We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events. These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene. Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells. Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells. All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion. Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.

Animals↗

PubMed1986

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

Base Sequence↗