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R Southgate

Publications and source records attributed to R Southgate.

At least 19 recordsLinked to original sources
PubMed2001

Alternative splicing of an amino-terminal PEVK-like region generates multiple isoforms of Drosophila projectin.

Drosophila projectin is an extremely large protein found within the muscle sarcomeric unit, parallel with the actin and myosin filaments. Projectin has been suggested as the elastic component of C-filaments in insect indirect flight muscles, which is consistent with its localization from the Z band to the tip of the A band in these muscles. Here, we describe the completion of the projectin sequence analysis, which defines projectin as a 1 MDa protein, composed of 39 immunoglobulin and 39 fibronectin III domains. This analysis led also to the identification of a domain rich in the amino acids P, E, V and K within the NH(2) terminus of projectin. The length of the projectin PEVK-like region varies from 100 to 624 amino acid residues, following a complex pattern of alternative splicing events. PEVK domains were first identified in vertebrate titin and they have been associated with the elasticity of the protein. The PEVK-like domain of the projectin isoforms in indirect flight muscles may contribute to the elastic function of the C-filaments. The synchronous projectin isoforms contain a PEVK-like region, and the possible non-elastic function(s) of this domain in synchronous muscles are discussed.

Alternative Splicing↗
PubMed2000

Drosophila projectin: a look at protein structure and sarcomeric assembly.

The large projectin protein is found in all Drosophila muscles; however, it shows a dual sarcomeric localization depending on the muscle type. In larval and adult synchronous muscles, projectin is found localized over the A-band. Initial in vitro binding assays indicate interactions of several projectin regions with themselves and myosin heavy chain. These interactions might be critical for the assembly of projectin over the myosin filament during embryonic myofibrillogenesis and larval growth. On the other hand, projectin localizes over the I-Z-I region in indirect flight muscles. Correspondingly, projectin is found in association with forming Z-bands during pupation and colocalizes with alpha-actinin and kettin.

Animals↗
PubMed1998

Structure of the Drosophila projectin protein: isoforms and implication for projectin filament assembly.

The protein composition of the various muscle types in Drosophila melanogaster has been studied quite thoroughly and the analysis has revealed many differences involving the usage of muscle specific isoforms of a given protein, as well as the presence of proteins restricted to one muscle type. Drosophila projectin, the giant protein component of the third filament is quite unusual as it not only shows specific isoforms in various muscle types, but these isoforms are located at different sarcomeric locations, I band in the IFM and A band in synchronous muscles. This may suggest distinct functions for the projectin protein in various muscles, as well as a different set of protein interactions for each projectin isoform. Projectin is encoded by a single gene and the isoforms were proposed to be the result of alternative splicing of a primary transcript. Here, we report the nearly complete sequence of Drosophila projectin, as well as the possible splicing patterns used to generate different isoforms. The overall domain organization in projectin is composed of repeated motifs I and II in a few specific patterns, similar to its Caenorhabditis homolog, twitchin. Sequence similarity between twitchin and projectin further suggests how some domains may possibly be important for protein interactions and/or functions. Alternative splicing operates at the COOH terminus, leading to a shorter projectin protein lacking some of the terminal motifs II and unique sequence. These isoforms are discussed in view of projectin differential size and localization.

Alternative Splicing↗
PubMed1996

Slow binding inhibition of phospho-N-acetylmuramyl-pentapeptide-translocase (Escherichia coli) by mureidomycin A.

Enzymes of the membrane cycle of reactions in bacterial peptidoglycan biosynthesis remain as unexploited potential targets for antibacterial agents. The first of these enzymes, phospho-N-acetylmuramyl-pentapeptide-translocase (EC 2.7.8.13), has been overexpresed in Escherichia coli and solubilized from particulate fractions. The work of W.A. Weppner and F.C. Neuhaus ((1977) J. Biol. Chem. 252, 2296-303) has been extended to establish a usable routine fluorescence-based continuous assay for solubilized preparations. This assay has been used in the characterization of the natural product, mureidomycin A as a potent slow binding inhibitor of the enzyme with Ki and Ki* of 36 nM and 2 nM, respectively.

Anti-Bacterial Agents↗
PubMed1996

Modes of action of tunicamycin, liposidomycin B, and mureidomycin A: inhibition of phospho-N-acetylmuramyl-pentapeptide translocase from Escherichia coli.

Using a continuous fluorescence-based enzyme assay, we have characterized the antibacterial agents tumicamycin and liposidomycin B as inhibitors of solubilized Escherichia coli phospho-N-acetylmuramyl-pentapeptide translocase. Tunicamycin exhibited reversible inhibition (Ki = 0.55 +/- 0.1 microM) which was noncompetitive with respect to the lipid acceptor substrate and competitive with respect to the fluorescent substrate analog, dansyl-UDPMurNAc-pentapeptide. Liposidomycin B exhibited slow-binding inhibition (Ki = 80 +/- 15 nM) which was competitive with respect to the lipid acceptor substrate and noncompetitive with respect to dansyl-UDPMurNAc-pentapeptide. These results provide insight into the molecular mechanisms of action of these two classes of nucleoside antibiotics.

Aminoglycosides↗
PubMed1995

Both synchronous and asynchronous muscle isoforms of projectin (the Drosophila bent locus product) contain functional kinase domains.

In Drosophila, the large muscle protein, projectin, has very different localizations in synchronous and asynchronous muscles, suggesting that projectin has different functions in different muscle types. The multiple projectin isoforms are encoded by a single gene; however they differ significantly in size (as detected by gel mobility) and show differences in some peptide fragments, presumably indicating alternative splicing or termination. We now report additional sequence of the projectin gene, showing a kinase domain and flanking regions highly similar to equivalent regions of twitchin, including a possible autoinhibitory region. In spite of apparent differences in function, all isoforms of projectin have the kinase domain and all are capable of autophosphorylation in vitro. The projectin gene is in polytene region 102C/D where the bentD phenotype maps. The recessive lethality of bentD is associated with a breakpoint that removes sequence of the projectin kinase domain. We find that different alleles of the highly mutable recessive lethal complementation group, l(4)2, also have defects in different parts of the projectin sequence, both NH2-terminal and COOH-terminal to the bentD breakpoint. These alleles are therefore renamed as alleles of the bent locus. Adults heterozygous for projectin mutations show little, if any, effect of one defective gene copy, but homozygosity for any of the defects is lethal. The times of death can vary with allele. Some alleles kill the embryos, others are larval lethal. These molecular studies begin to explain why genetic studies suggested that l(4)2 was a complex (or pseudoallelic) locus.

Amino Acid Sequence↗
PubMed1990

Synthesis and antibacterial activity of C-2 alkenylthio-carbapenem derivatives.

A number of C-2 aminoalkenylthio-carbapenem derivatives possessing both the (5R,6R, 8S)- and the (5R,6S,8R)-stereochemistries have been prepared from the olivanic acids MM 22382 and MM 22383, respectively. Certain members of this new class of compounds displayed potent, broad spectrum antibacterial activity as well as improved stability to human kidney homogenate.

Bacteria↗
PubMed1990

Synthesis and antibacterial activity of C-2 carboxyethenylthio-carbapenem derivatives.

A number of C-2 carboxyethenylthio-carbapenem derivatives possessing either the (5R,6R,8S)- or the (5R,6S,8R)-stereochemistries have been prepared from the olivanic acids MM 22382 (1) and MM 22383 (4), respectively. Their in vitro antibacterial activities and stabilities to human kidney homogenate are superior to those of the parent compounds, particularly in the latter series.

Animals↗
PubMed1985

Nucleotide sequences responsible for the thermal inducibility of the Drosophila small heat-shock protein genes in monkey COS cells.

The promoter regions of the Drosophila melanogaster small heat-shock protein genes have been analysed in order to localize those sequences responsible for their heat-shock transcriptional inducibility. Different lengths of the 5' DNA sequences of these four genes were each fused individually to the Herpes simplex virus thymidine kinase (HSV-tk) transcription unit. These hybrid genes were constructed in a simian virus 40 recombinant vector for transfection in permissive monkey COS cells and tested for their heat-shock inducibility. The hsp22/HSV-tk and hsp26/HSV-tk fusion genes were found to be heat-inducible at 43 degrees C, giving rise to correctly initiated transcripts, but transcriptionally quiescent at 37 degrees C (control temperature). The hsp23 and hsp27 fusion gene constructs are, however, not heat-shock-inducible; no transcripts being detectable from hsp27/HSV-tk constructs at either temperature and all hsp23/HSV-tk clones being faithfully but constitutively expressed at low levels at both temperatures. By testing a series of 5' deletion mutants in hsp22/HSV-tk, a homologous sequence located adjacent to the TATA box in both the hsp22 and hsp26 genes was identified as being responsible for their heat-shock activation. This control element corresponds to the Pelham "consensus sequence", previously described for the Drosophila hsp70 genes. The possible modes of transcriptional induction of all four genes are discussed.

Animals↗
PubMed1985

Mutual pro-drugs of the olivanic acids and renal dipeptidase inhibitors.

The carbapenem antibiotics, which include the olivanic acids and thienamycin, possess potent broad spectrum antibacterial activity. They are however extensively metabolized by the renal dipeptidase enzyme, dehydropeptidase I. As a result of this degradation, only low urinary recoveries of antibiotic are obtained in vivo. The preparation of mutual pro-drugs of the olivanic acids and an inhibitor of the renal dipeptidase enzyme is described. MM 22382 and MM 13902 have been combined with Z-2-isovaleramidobut-2-enoic acid as double esters of formaldehyde hydrate. Administration of these linked esters to mice results in improved urinary recoveries of antibiotic.

Amides↗
PubMed1983

Nucleotide sequence analysis of the Drosophila small heat shock gene cluster at locus 67B.

The four small heat shock protein genes of Drosophila melanogaster clustered at cytological locus 67B have been characterized by DNA sequencing. Over 6250 nucleotides, covering the 5', protein-coding and 3' regions of these genes have been determined together with their predicted amino acid sequences. Each gene possesses characteristic eukaryotic 5' and 3' sequence elements and a single uninterrupted protein-coding region. The four encoded polypeptides of 19,700, 20,600, 23,000 and 23,600 Mr share a homologous stretch of 108 amino acid residues, representing 51 to 62% of their lengths. This region is flanked by sequences of dissimilar length and amino acid composition, located mainly at the amino-terminal end, but also at the extreme carboxyl termini of these proteins. The first 14 amino acids exhibit a small degree of homology, both amongst themselves and with some signal peptides and a transmembrane protein. Investigation of the hydrophilic/hydrophobic characteristics of the four polypeptides revealed, within the conserved 108 amino acid stretch, the presence of an alpha-helical region of very prominent local hydrophilicity, which probably represents a surface structural domain common to each protein. Sequence analysis with respect to transcription initiation and termination and possible regulatory signals is discussed together with some structural predictions for the four proteins.

Amino Acid Sequence↗
PubMed1983

Comparative biological properties of some synthetic olivanic acid analogues.

A series of olivanic acid/thienamycin analogues have been prepared by total synthesis. Particular attention was given to the effect of the side-chain substituents on the chemical, beta-lactamase and metabolic stability of the final products. All of the compounds possessed a broad and high level of in vitro antibacterial activity against Gram-positive and Gram-negative organisms including beta-lactamase-producing strains. Two derivatives (8c) and (8j) were selected for further evaluation on the basis of in vitro activity, ease of synthesis and stability parameters. The improved metabolic stability of the selected analogues, relative to the naturally-occurring olivanic acid, MM 13902, could be demonstrated in terms of better activity, higher blood levels and improved urinary recovery in in vivo studies in mice.

Animals↗