Biomedical subjects

T D Ingolia

Publications and source records attributed to T D Ingolia.

At least 19 recordsLinked to original sources

PubMed1997

The therapeutic potential of inducing molecules.

Many fundamental developmental mechanisms that result in the generation and formation of specific cells and organ systems are not limited to embryogenesis. Regenerative processes in the adult serve to help regulate cellular turnover in systems where specific cell numbers or balance is crucial. In addition, these processes can be re-activated to replace cell populations which are lost due to damage or disease. Induction of differentiation of one cell type by another is one mechanism that also acts as a regenerative process in the adult. Molecules which direct these processes are termed inducing molecules and they are prevalent and active in many adult organ systems such as the haematopoietic, osteogenic and reproductive systems. To date, recombinant forms of inducing molecules identified in the haematopoietic system are being used clinically to stimulate production of specific cell types that are depleted during chemotherapy or dialysis treatments. The prospect that other inducing molecules for other organ systems may be as effective clinically has prompted intense research. The ability to harness inducing molecules as effective therapeutic agents to treat cell deficits in congenital or acquired disease states is the goal of several biotechnology companies.

Journal Article↗

PubMed1989

Active bacterial luciferase from a fused gene: expression of a Vibrio harveyi luxAB translational fusion in bacteria, yeast and plant cells.

The luxA and luxB genes encoding the luciferase from Vibrio harvey were fused by site-directed mutagenesis so that one polypeptide was encoded by the fused gene. The fused gene facilitated light production in Escherichia coli, Saccharomyces cerevisiae, and Nicotiana plumbaginifolia when the substrates decanal and reduced flavin mononucleotide were present. The specific activity of the encoded enzyme is not known. In E. coli and S. cerevisiae cells the light emission could be measured in viable, intact cells. The luxAB fusion provides a simple reporter gene for in vivo measurement of promoter strength in these species and may be useful in other systems as well.

Base Sequence↗

PubMed1989

Cloning and characterization of beta-lactam biosynthetic genes.

Seven genes coding for two different enzymes of the penicillin/cephalosporin biosynthetic pathway have been cloned from fungal and bacterial sources. Using amino acid sequences derived from the purified enzymes, oligonucleotide probes were designed to hybridize to their cognate genes in a genomic library. The high degree of similarity (57%) between enzymes of bacterial and fungal origin suggests a horizontal transfer of a primordial beta-lactam pathway, probably from a bacterial cell to a fungal cell. Overproduction of the proteins in Escherichia coli has allowed further study of the mechanism of action of these important enzymes.

Amino Acid Sequence↗

PubMed1989

Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase.

Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.

Amino Acid Sequence↗

PubMed1988

Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins.

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.

Amino Acid Sequence↗

PubMed1988

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.

Amino Acid Sequence↗

PubMed1987

Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis.

The predicted amino acid sequences of isopenicillin N synthetase from both Cephalosporium acremonium and Penicillium chrysogenum have two cysteine residues in analogous positions (Cys-106 and Cys-255 in the C. acremonium numbering). To examine the role of these cysteine residues in the activity of the C. acremonium enzyme, we used site-directed in vitro mutagenesis to change these cysteine residues to serine residues. Mutation of Cys-255 reduces specific activity approximately equal to 50%, whereas mutation of Cys-106 or mutation of both Cys-106 and Cys-255 reduces specific activity about 97%. This suggests that the cysteines are important but not essential for IPNS activity. Alkylation of IPNS also almost completely inactivated the enzyme, but residual activity could have been due to incomplete alkylation. Atomic substitution via genetic manipulation in this case is a more accurate means of assessing the role of sulfhydryl moieties in enzyme activity.

Acremonium↗

PubMed1986

Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum.

The isopenicillin N synthetase (IPS) gene from Penicillium chrysogenum was isolated from a recombinant bacteriophage lambda library using the Cephalosporium acremonium IPS (cIPS) gene as a heterologous hybridization probe. The protein coding region of the P. chrysogenum IPS (pIPS) gene was about 74% homologous to the cIPS gene, and the predicted amino acid sequences of the encoded proteins were about 73% homologous. Escherichia coli cells with the pIPS gene contained IPS activity whereas untransformed cells were completely devoid of this enzymatic activity. The transformed cells were also shown to contain an abundant protein accounting for about 10% of total cell protein which reacted strongly with anti-cIPS antiserum.

Acremonium↗

PubMed1984

Plasmid-mediated complementation of a delta-aminolevulinic-acid-requiring Saccharomyces cerevisiae mutant.

Recombinant plasmids able to complement the Saccharomyces cerevisiae ole3 mutation were isolated. The nucleotide sequence responsible for complementation was localized to a 3.5-kb region. The level of delta-aminolevulinate (ALV) synthase activity in wild-type cells was six-fold lower than in plasmid-transformed ole3 mutant cells. Certain clones secreted a compound that supported growth of a lawn of adjacent ole3 mutant cells.

5-Aminolevulinate Synthetase↗

PubMed1984

Stimuli that induce a yeast heat shock gene fused to beta-galactosidase.

Saccharomyces cerevisiae contain a multigene family related to the Drosophila heat shock gene hsp70. Two members of this family, YG100 and YG101, have been previously characterized (Ingolia et al., Mol. Cell. Biol. 2:1388-1398, 1982), and only YG100 was found to have elevated levels of transcription after heat shock. The yeast hsp70 genes contained on YG100 and YG101 were truncated and fused to the Escherichia coli lacZ gene contained on pMC1587 (Casadaban et al., Methods Enzymol. 100:283-308, 1983). The resulting plasmids directed synthesis of the beta-galactosidase gene as measured by in vitro enzyme assays and by colorimetric assays on plates. The expression level from the YG101 gene was constant under all the conditions tested, whereas expression driven by the YG100 gene could be induced over 50-fold. Other stimuli besides heat, including recovery from anoxia and high cell density, were found to strongly induce YG100 gene expression. Most physical and chemical stimuli tested, including UV irradiation, zymolyase treatment, and ethanol, did not stimulate expression of this heat shock gene.

Culture Media↗

PubMed1983

In vitro transcription of Drosophila actin and 70,000-dalton heat shock protein genes.

Drosophila genomic DNAs containing a chromosomal locus 87C1 70,000-dalton heat shock protein gene, the locus 79B actin gene, and the 88F actin gene have been used as templates in an in vitro HeLa transcription system. RNA polymerase II-dependent transcription initiates from specific sites on the heat shock protein gene and the 79B actin gene. The locations of the transcription start sites were determined by two types of experiments: sizing of RNA runoff transcripts and S1 nuclease mapping of the 5' terminus of the in vitro transcripts. Transcription initiates at or near the in vivo initiation site of the heat shock protein gene and initiates at or near a site 14 nucleotides downstream of the in vivo start site of the 79B actin gene. The addition of the 79B actin, 88F actin, or heat shock protein templates to a HeLa extract transcription reaction precluded the transcription of a second template subsequently added. The exclusion occurs rapidly, within 15 s, is not dependent on transcription, and is only partially resistant to high concentrations of the second added template. We propose that stable protein-promoter complexes play an important role in maintaining exclusive transcription of the first template added in vitro.

Actins↗

PubMed1983

Analysis of a bacterial hygromycin B resistance gene by transcriptional and translational fusions and by DNA sequencing.

We have characterized hygromycin B and apramycin resistance genes from an E. coli plasmid. We have localized the coding and control regions of these genes by deletion of DNA fragments from plasmids containing the genes. It was found that polypeptides with apparent molecular weights of 33,000 and 31,500 daltons are encoded by the apramycin resistance gene and polypeptides with apparent molecular weights of 42,500 and 41,500 daltons are encoded by the hygromycin B resistance gene. DNA sequence analysis identified a typical promoter sequence upstream of the genes. Deletion of this promoter eliminated both resistance phenotypes, and hygromycin B resistance could be restored by substitution of a promoter from a foreign gene. The region known to be necessary for hygromycin B resistance contained an open reading frame large enough to encode the hygromycin B resistance gene product. This open reading frame was fused with the amino terminus of beta-galactosidase. This hybrid gene conferred hygromycin resistance to E. coli, and expression of resistance was under IPTG control.

Anti-Bacterial Agents↗

PubMed1983

Expression of Drosophila heat-shock cognate genes during heat shock and development.

The Drosophila genome contains a number of genes related to the major heat-shock-inducible (Hsp70) genes of Drosophila. The structure and expression of three of these heat-shock cognate (Hsc) genes have been studied; Hsc1 (previously described), Hsc2, and Hsc4 are dispersed on chromosome 3 at cytological loci 70C, 87D, and 88E, respectively. RNA homologous to Hsc4 was shown by hybridization selection translation experiments to encode a protein of approximately 70kd, with similar, but distinguishable, mobility to that of the heat-shock-inducible 70K protein. Because of variance in size of the 5' nonprotein coding region, it was possible to identify in adult flies distinct transcripts from each gene. Unlike transcripts from Hsp genes, the abundance of the Hsc transcripts was not increased after heat shock. Transcription of these Hsc genes is regulated during development. While transcripts from Hsc4 were equally abundant in RNA isolated from embryos, larvae and adults, Hsc1 and Hsc2 transcripts were not detected in embryo and larval RNA, and are therefore at least 20 times less abundant in these stages than in adults. The primary DNA sequences of the regions homologous to that encoding the amino terminus of hsp70 were compared. Approximately 76% homology of the base sequence and 78% homology of the deduced amino acid sequence were found among the four genes. The Hsp70 genes contain no intervening sequences; Hsc4 contains no insertions relative to Hsp70 in the region encoding the first 101 aa. However, in the codon specifying aa66 Hsc1 is interrupted by 1.7 kb of DNA; in the codon for aa55 Hsc2 contains an insertion of about 650 b. The sequences at the 5' and 3' junctions of the insertions in both Hsc1 and Hsc2 are similar to those found in intervening sequences of Drosophila and of many other organisms.

Amino Acid Sequence↗

PubMed1982

Drosophila gene related to the major heat shock-induced gene is transcribed at normal temperatures and not induced by heat shock.

A gene related to the major heat shock-induced (hsp70) gene of Drosophila has been isolated from the sibling species D. melanogaster and D. simulans. This heat shock-cognate (hsc70) gene is present at cytological locus 70C. The primary sequence of approximately one-third of the protein-coding region has been compared with that of the hsp70 gene; 72% homology of the base sequence and 74% homology of the deduced amino acid sequence was found. In the codon specifying amino acid 66, the hsc70 gene contains an insertion of 1.7 kilobases; the hsp70 genes contain no intervening sequences. The sequence at the 5' and 3' junctions of the insertion is similar to that found in many intervening sequences. cDNA extension experiments indicate that the hsc70 gene is transcribed at normal temperatures in adult flies and that transcription is not enhanced by heat treatment.

Amino Acid Sequence↗

PubMed1982

Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin.

The primary base sequence of the protein coding regions of the four small heat shock genes of Drosophila melanogaster present at cytological locus 67B has been determined. A single open reading frame large enough to encode a small heat shock protein is found for each gene. The molecular weights of the predicted proteins are in good agreement with experimentally determined values obtained from gel electrophoresis. The predicted amino acid sequences of the four small heat shock genes show striking homologies over approximately 50% of their lengths. This region of extensive homology extends from about amino acid 85 to amino acid 195 out of a total of approximately 200 amino acids. Comparison of the predicted sequence with the known sequences of other proteins revealed a remarkable similarity between this region of homology and the corresponding region of mammalian alpha-crystallin. The possible functional significance of this structural similarity is discussed.

Amino Acid Sequence↗

PubMed1982

Saccharomyces cerevisiae contains a complex multigene family related to the major heat shock-inducible gene of Drosophila.

Saccharomyces cerevisiae contains a family of genes related to the major heat shock-induced gene of Drosophila (hsp 70). Two members of the multigene family (YG100 and YG101) were isolated. The primary DNA sequences of more than one-half of the protein-encoding regions of YG100 and YG101 were determined and compared with the Drosophila hsp 70 gene sequence; the predicted amino acid sequences were 72 and 64% homologous to the sequence of the Drosophila hsp 70 protein, respectively. The predicted amino acid sequences of the yeast genes were 65% homologous. Our results demonstrate a striking sequence conservation of hsp 70-related sequences in evolution. Hybridization of the S. cerevisiae genes to total S. cerevisiae DNA indicated that the multigene family consists of approximately 10 members. Hybridization of labeled RNAs from heat-shocked and control cells suggested that, like transcription of the Drosophila hsp 70 gene, transcription of YG100 or a closely related gene is enhanced after heat shock. However, the amount of RNA sequences homologous to YG101 was reduced after heat shock. A multigene family related to the hsp 70 gene exists in Drosophila; transcription of some members is induced by heat shock, whereas transcription of others is not. Our results suggest that S. cerevisiae, like Drosophila, contains a multigene family of hsp 70-related sequences under complex transcriptional regulation and that the differential control, as well as the nucleotide sequence, has been highly conserved in evolution.

Amino Acid Sequence↗

PubMed1981

Primary sequence of the 5' flanking regions of the Drosophila heat shock genes in chromosome subdivision 67B.

The 5' flanking regions of the four small heat shock genes of Drosophila melanogaster from cytological locus 67B have been characterized. Approximately 500 bp of the primary sequence upstream from the proposed site of initiation of translation has been determined and the 5' end of the messenger RNAs have been localized for each gene. Each of the four genes contains an A-T rich sequence, either TATAAATA or TATAAAAG, which is flanked by a G-C rich region. This A-T rich sequence, which ends about 23 bp upstream from the proposed site of initiation of transcription, is similar to those found in most eukaryotic genes. Novel features of these four genes include a region of homology beginning near the proposed site of initiation of transcription and extending about 20 bp into the 5' noncoding region of the genes. This sequence is also found in the gene for the major heat shock protein, hsp 70. The leaders of these five heat shock genes are long, from 111 to 253 bases in length, as well as unusually A rich, from 46% to 51% A. In addition, each of the four small genes contains the sequence ACTTTNA, 195 +/- 12 bp from the proposed site of initiation of transcription.

Animals↗

PubMed1980

Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions.

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5' end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5' flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upstream from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24-30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.

Amino Acid Sequence↗