Biomedical subjects

S Ponnambalam

Publications and source records attributed to S Ponnambalam.

29 records · Page 2Linked to original sources

PubMed1994

Chromosomal location and some structural features of human clathrin light-chain genes (CLTA and CLTB).

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to be encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.

Animals↗

PubMed1994

The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network.

The membrane-spanning and cytoplasmic domains of CD4 and CD8 were replaced by those of TGN38. After transient expression in HeLa cells, the location of the hybrid proteins was determined using immunofluorescence and quantitative immuno-electron microscopy, FACS analysis and metabolic labeling. The membrane-spanning domain was found to contain a signal that localized hybrid proteins to the TGN. This was in addition to the signal previously identified in the cytoplasmic domain (Bos, K., C. Wraight, and K. Stanley. 1993. EMBO (Eur. Mol. Biol. Organ) J. 12:2219-2228. Humphrey, J. S., P. J. Peters, L. C. Yuan, and J. S. Bonifacino. 1993. J. Cell Biol. 120:1123-1135. Wong, S. H., and W. Hong. 1993. J. Biol. Chem. 268:22853-22862). The different properties of these two signals suggest that each operates by a different mechanism.

Amino Acid Sequence↗

PubMed1993

Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator.

The ypt/rab proteins are a family of small GTP-binding proteins thought to be required for different stages of membrane traffic. From the fission yeast Schizosaccharomyces pombe we have isolated and characterized ypt5, a gene encoding a homologue of rab5, a mammalian protein apparently involved in regulating fusion of early endosomes. Recombinant ypt5 protein bound GTP. The ypt5 gene was found to be essential for viability on minimal media, but ypt5-disrupted cells grew slowly on some rich media and accumulated a population of small vesicles not observed in wild-type cells. Canine rab5 cDNA could replace the ypt5 gene in S. pombe and restore normal growth and viability. Ypt5 protein expressed in mammalian cells colocalized with the transferrin receptor to early endosomes. Thus, molecular aspects of the early endocytic pathway may be conserved between mammalian cells and S. pombe and hence may be amenable to genetic analysis.

Amino Acid Sequence↗

PubMed1991

Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics.

Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain.

Amino Acid Sequence↗

PubMed1990

Conservation and diversity in families of coated vesicle adaptins.

The complete sequence of the beta adaptin subunit of the plasma membrane adaptor complex from coated vesicles has been elucidated. Complementary cDNA clones from human fibroblasts, rat lymphocytes, and bovine lymphocytes have been isolated, sequenced, and compared with each other and with beta adaptin sequences from rat brain (Kirchhausen, T., Nathanson, K.L., Matsui, W., Vaisberg, A., Chow, E.P., Burne, C., Keen, J.H., and Davis, A.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2612-2616). Surprisingly, the 937-amino acid beta adaptin polypeptide is totally conserved between species. This remarkable homology contrasts with the absence of significant sequence similarity between the alpha (Robinson, M.S. (1989) J. Cell Biol. 108, 833-842) and beta adaptins of the plasma membrane adaptor complex. Diversity within each adaptin family is created by the expression of different genes and by tissue-specific differential splicing. The structures of the beta and alpha adaptins can both be divided into two globular domains interconnected by a variable and potentially flexible stalk domain.

Adaptor Protein Complex alpha Subunits↗

PubMed1988

Functional analysis of different sequence elements in the Escherichia coli galactose operon P2 promoter.

Starting with a DNA fragment containing the galactose operon P2 promoter, we made a series of deletions that progressively replaced DNA sequences upstream of the transcription startpoint and determined their effects on P2 activity. The results show that specific sequences upstream of -32 are not important. Removal of the sequence 5'-CACA-3' from -32 to -28 reduces P2 activity by 50%: longer deletions to -16 further reduce activity but do not remove the information specifying the transcription startpoint. DNA sequences between -32 and -16 at gal P2 assist the isomerization of RNA polymerase from closed to open complexes rather than contributing to the initial binding of RNA polymerase. The activity of gal P2 in the absence of -35 region sequences is dependent on the sequence TG just upstream of the -10 hexamer, TATACT: a mutation at -14 changing the TG sequence to TT totally inactivates P2. However, P2 activity can be restored if the consensus -35 region sequence TTGACA is cloned 17 bp upstream of the -10 hexamer. Thus, for transcription initiation, the -10 hexamer, TATACT, must 'cooperate' with upstream sequences that may be located either around -35 or -14.

Base Sequence↗

PubMed1987

Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters. Interactions with RNA polymerase and the cyclic AMP receptor protein.

We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, S1 and S2. In the presence of this point mutation, transcription initiates at a third start point 14/15 bp downstream of S1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to P1. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.

DNA-Directed RNA Polymerases↗

PubMed1987

Binding of Escherichia coli RNA polymerase to a promoter carrying mutations that stop transcription initiation.

The gal P2 promoter can be inactivated by point mutations located in the -10 hexamer sequence or immediately upstream from it. Mutations at either site reduce expression in vivo and prevent the formation, in vitro, of tight complexes with RNA polymerase that give a strong footprint and can initiate transcription. However, with a mutation upstream from the -10 region, RNA polymerase could still make a specific contact with gal promoter DNA as judged by interference with cleavage by restriction enzyme SfaNI at a site within the promoter. In contrast, with a mutation in the -10 hexamer sequence, RNA polymerase could not make this contact and does not interfere with restriction by SfaNI.

Base Sequence↗

PubMed1987

RNA polymerase molecules initiating transcription at tandem promoters can collide and cause premature transcription termination.

Using purified E. coli RNA polymerase we have studied the transcription in vitro of a series of DNA fragments carrying two tandemly arranged promoters, where the corresponding transcription start points were separated by 263, 138, 83 and 78 base pairs. In the case where the transcription start points are 83 base pairs apart, there is an interaction between RNA polymerase molecules transcribing from the two promoters. This interaction results in premature termination of the upstream transcript at a precise site. We propose that this is the result of RNA polymerase transcribing from the upstream promoter bumping into polymerase at the downstream promoter. The interaction between the two polymerase molecules is crucially dependent on the distance between the two promoters.

DNA-Directed RNA Polymerases↗

PubMed1986

Transcription initiation at the Escherichia coli galactose operon promoters in the absence of the normal -35 region sequences.

The gal operon regulatory region contains two overlapping promoters, P1 and P2, regulated by cyclic AMP and the cyclic AMP receptor protein (cAMP X CRP). Starting with a mutation that eliminated P1, the promoter that is usually dependent on cAMP X CRP, we constructed a series of deletions that substituted increasing amounts of DNA sequence from upstream of P2, the promoter that usually functions in the absence of cAMP X CRP. Expression from P2 in vivo was halved by deletions that replace the -35 region with unrelated sequences, showing that the -35 sequence participates in promoter function, but is not essential. In vitro studies show that replacement of the -35 sequence increases the time for open complex formation at P2, but does not alter the transcription start point. We examined the effects of the same deletions at the wild type gal promoter region: again, the deletion that replaces the -35 region halves expression in vivo. However, in this case, in the absence of cAMP X CRP, the deletion switches expression from the P2 promoter to P1, the promoter that is usually dependent on cAMP X CRP. Moreover, although the deletion also removes the specific cAMP X CRP binding site, this P1 activity is sharply inhibited in a crp+ background. We argue that this is due to a direct contact between CRP and RNA polymerase bound at the P1 Pribnow box, and we discuss the role of the -35 sequence at these and other promoters.

Base Sequence↗

PubMed1986

Mutations that reduce expression from the P2 promoter of the Escherichia coli galactose operon.

We describe the isolation and characterisation of twelve different mutations that reduce gene expression from the galP2 promoter, starting with a gal regulatory region with a mutation that inactivated galP1, the cAMP-CRP-dependent promoter. Seven of the new mutations reduce the initiation of transcription at P2 whereas the others reduce translation initiation of the first gal operon gene, galE. Two of the mutations affecting translation fall in the galE initiation codon and the Shine-Dalgarno sequence. Mutations that allow the formation of a stem-loop structure in the messenger including this sequence also reduce translation. A deletion of 11 bp, upstream of the Shine-Dalgarno sequence, almost totally prevents translation. Although none of the point mutations that reduced transcription initiation at P2 fall in the -35 region, we repeatedly isolated insertions in this zone. The point mutations all fell around the -10 region: the strongest effects were found with mutations that altered the sequence away from the consensus that has been established for Escherichia coli promoters. The effects of the two strongest P2 mutations were investigated in the absence of the P1 mutation used for their isolation. One mutation, a T:A to C:G transition at -12, inactivates both P2 and P1. In contrast the other, a T:A to G:C transversion at -19, specifically inactivates P2, but leaves P1 partially active even in the absence of cAMP-CRP. The implications of this are discussed in the context of how cAMP-CRP controls the balance between transcription from P2 and P1 at the gal operon regulatory region.

Base Sequence↗