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G Heinrich

Publications and source records attributed to G Heinrich.

At least 73 records · Page 4Linked to original sources

An improved algorithm for nucleic acid secondary structure display.

An improved algorithm for the display of nucleic acid secondary structures is presented. It is particularly suitable for large sequence segments and it automatically generates an aesthetically pleasing display of the structure with very limited overlap of strands. Structural similarities in different structures are conserved in the display thus greatly aiding structural homology comparisons. Using the algorithm, we illustrate the effect of ribosome translocation on the secondary structure of a rat neuropeptide messenger RNA.

Algorithms↗

Genes encoding proteins with homologous contiguous repeat sequences are highly expressed in the serous cells of the rat submandibular gland.

An abundant class of secreted salivary polypeptides is characterized by the presence of identical and contiguous repeats of amino acid sequences within the polypeptide chains, and includes the proline-rich proteins. We discovered a new family of contiguous repeat polypeptides (CRPs) that is related to the proline-rich proteins but contains little proline. Analysis of salivary mRNAs and liver DNA by molecular cloning, DNA sequence determinations, and Northern and Southern blot hybridization revealed several closely related CRP mRNAs and at least 10 CRP-related genes. We further analyzed two CRP mRNAs of 850 and 920 nucleotides and the gene encoding the larger CRP mRNA. The two mRNAs contain the same 69-base repeats in their coding regions and are identical in their 5'- and 3'-untranslated tracts. However, they differ in the number of contiguous repeats (four versus five) and a segment at the 3' end of the coding region which encodes closely related but unique COOH termini of the CRPs. These structural features suggest a recent gene conversion. The CRP gene analyzed is divided into three exons that encode (i) 5'-untranslated tract and signal sequence, (ii) secreted polypeptide, and (iii) 3'-untranslated tract, respectively. CRP mRNA contains two open reading frames. The longer open reading frame encodes a CRP precursor with a signal sequence of 17 amino acids, four to five contiguous repeats of 23 amino acids, and a variable COOH region that begins with two segments related to the contiguous repeats. Immunochemical analysis of salivary gland slices with antisera raised against peptides corresponding to two regions of the larger open reading frame revealed intense staining only of the serous cells of the submandibular glands. 35S-Labeled oligonucleotides complementary to CRP mRNA specifically hybridized to the same cells.

Amino Acid Sequence↗

Regulation of nerve growth factor gene expression in primary brain monolayer cultures: effects of dibutyryl cyclic AMP and sodium butyrate.

Primary brain cultures composed of a heterogeneous population of cells, most of which reacted with anti-fibronectin and only few with anti-GFAP antiserum, were found to synthesize a mRNA indistinguishable from mouse submandibular gland nerve growth factor (NGF) mRNA in hybridization characteristics and electrophoretic behavior in agarose gels. The levels of this mRNA were increased 2.5-10 fold by 1 mM dibutyryl cyclic AMP (db-cAMP) and sodium butyrate within 24 h, but not by 10 microM forskolin, and were unaffected by serum in the culture medium. These observations demonstrate that brain cells synthesize a NGF mRNA in primary culture and that the butyrate moiety of db-cAMP enhances NGF gene expression in these cells, probably by a modification of chromatin structure in and around the NGF gene.

Animals↗

Neuropeptide Y gene expression in PC12 cells and its regulation by nerve growth factor: a model for developmental regulation.

Neuropeptide Y (NPY) is a 36 amino acid neuronal peptide which has previously been described within a subset of sympathetic neurons. The presence of the messenger RNA that encodes NPY (NPY mRNA) has been studied in the PC12 cell line, as this is commonly used as a model of neuronal differentiation. Low levels of the NPY mRNA were identified in resting naive cells. Levels were induced five-fold by the addition of nerve growth factor to the medium at a dose of 30 ng/ml. Lower doses of nerve growth factor had no effect on NPY mRNA. The effect was rapid in onset being apparent within 12 h and near maximal at 24 h. A smaller two-fold increase in NPY mRNA was observed in cells treated with epidermal growth factor (3 ng/ml) over the same time course but no effect was observed with fibroblast growth factor (3 ng/ml), bradykinin (10(-6) M) or dexamethasone (10(-6) M). These results indicate that NPY gene expression is regulated in PC12 cells at the level of NPY mRNA.

Animals↗

Molecular structure of mammalian neuropeptide Y: analysis by molecular cloning and computer-aided comparison with crystal structure of avian homologue.

Identification and characterization of the cDNA encoding rat neuropeptide Y revealed the nucleotide sequence coding for a 98-amino acid precursor. The deduced amino acid sequence for rat neuropeptide Y is identical to the human peptide and is highly homologous to avian pancreatic polypeptide. The tertiary structure of avian pancreatic polypeptide has been previously derived from crystallographic data by Blundell and coworkers. The homology between neuropeptide Y and avian pancreatic polypeptide preserves all of the residues essential for the maintenance of the tertiary structure. Thus, it has been possible to compute a three-dimensional model of the mammalian neuropeptide, neuropeptide Y, based on the known structure of the avian homologue. This model suggest that neuropeptide preserves a compact tertiary structure characterized by extensive hydrophobic interactions between an N-terminal polyproline-II-like helix and a C-terminal alpha-helix. The model has been used to identify amino acids residing in key positions within this structure and, thereby, to direct future analysis of neuropeptide Y structure-function relationships.

Amino Acid Sequence↗

Mapping polypeptide hormone genes in the mouse: somatostatin, glucagon, calcitonin, and parathyroid hormone.

Mouse-Chinese hamster hybrids segregating mouse chromosomes were analyzed by Southern hybridization techniques to map the genes for somatostatin (Smst), glucagon (Gcg), calcitonin (Calc), and parathyroid hormone (Pth). The mouse gene for somatostatin, detected on a 20-kb EcoRI fragment, is located on mouse chromosome 16. Glucagon cDNA hybridized to a 14-kb EcoRI fragment residing on chromosome 2. Calcitonin and parathyroid hormone genes, detected on 7.8-kb HindIII and 6.0-kb BamHI fragments, respectively, were on mouse chromosome 7. The calcitonin and parathyroid hormone genes appear to be part of a larger linkage group which has been conserved in mouse and man.

Animals↗

Cortisone, testosterone, and aldosterone reduce levels of nerve growth factor messenger ribonucleic acid in L-929 fibroblasts.

Previous studies have shown that steroid hormones reduce concentrations of nerve growth factor (NGF) in medium conditioned by L-929 fibroblasts (L cells). In this study, we extend those observations and have measured in L cells the effects of hormone treatment on mRNA encoding NGF. L Cells were grown for 3 days in the presence or absence of hormones. NGF in conditioned medium was measured by NGF RIA; NGF mRNA was measured in cell extracts by Northern blot analysis. Cortisone reduced NGF levels in conditioned medium below the limit of detection of the RIA (less than 10% of control values) with an ED50 of 5 X 10(-9) M; NGF mRNA was reduced to 12% of control levels with an ED50 of 1 X 10(-8) M. Reductions in mRNA were maximal within 3 h and were completely reversed 12 h after removal of the hormone. Levels of NGF in conditioned medium were also undetectable in cultures treated with testosterone, and mRNA levels were reduced by 80%; the ED50 for both effects was 4 X 10(-9) M. Aldosterone (1 X 10(-6) M) reduced NGF to below detectable levels and NGF mRNA by 70%. Progesterone and thyroid hormone had no effect on NGF or NGF mRNA. 17 beta-Estradiol reduced levels of NGF in medium by 50%, but had no detectable effect on levels of NGF mRNA. These results suggest that cortisone, testosterone, and aldosterone decrease NGF levels in L cell-conditioned medium by reducing the cellular content of NGF mRNA.

Aldosterone↗

Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing.

Glucagon is a pancreatic hormone of 29 amino acids that regulates carbohydrate metabolism and glicentin is an intestinal peptide of 69 amino acids that contains the sequence of glucagon flanked by peptide extensions at the amino and carboxy termini. The glucagon gene encodes a precursor containing glucagon and two additional, structurally related, glucagon-like peptides separated by an intervening peptide. These peptides are encoded in separate exons. To determine whether the pancreatic and intestinal forms of glucagon arise by alternative RNA and/or protein processing, we used antisera to synthetic glucagon-like peptides and exon-specific, complementary oligonucleotides for analyses of proteins and mRNAs in pancreatic and intestinal extracts. Preproglucagon mRNAs are identical, but different and highly specific peptides are liberated in the two tissues. Immunocytochemistry shows colocalization of glucagon and the two glucagon-like peptides in identical cells. We conclude that diversification of preproglucagon gene expression occurs at the level of cell-specific post-translational processing.

Amino Acid Sequence↗

Nerve growth factor mRNA in brain: localization by in situ hybridization.

Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons.

Animals↗

Genes for insulin I and II, parathyroid hormone, and calcitonin are on rat chromosome 1.

Insulin, parathyroid hormone, and calcitonin are polypeptide hormones that regulate important physiological processes in target tissues. Rat genes encoding each hormone were chromosomally assigned to rat chromosome 1. Both rats and mice have two insulin genes (I and II). However, in contrast to mice in which insulin I and II are asyntenic, rat insulin I and II were both localized to chromosome 1. This study identifies a conserved syntenic group on rat chromosome 1, and implies that mouse insulin I and II genes were chromosomally separated after rats and mice diverged 20-35 million years ago.

Animals↗

Anti-thyroid peroxidase antibody in patients with autoimmune thyroid disease: possible identity with anti-microsomal antibody.

Thyroid microsomal antigen and peroxidase (TPO) have a close intracellular anatomical relationship, especially in exocytotic vesicles. We considered that antibodies to microsomal antigen might react with TPO and therefore looked for the presence of antibodies against TPO in the serum of patients with autoimmune thyroid disease (AITD). TPO was prepared from Graves' thyroid glands, solubilized by n-octyl glucoside, and its activity was assayed by the guaiacol method. Control sera and sera with a positive microsomal hemagglutination test (MCHA(+) ) were assayed for their ability to precipitate TPO activity by incubation of sera with TPO and protein A. We identified MCHA(+) sera which caused precipitation of TPO activity, and the extent of precipitation was related to the amount of serum added. A significant correlation was present between this anti-peroxidase activity and microsomal antibodies titers, measured by a micro-ELISA method. Affinity columns prepared from immunoglobulins of MCHA(+) sera, coupled to Reacti-Gel (6X), bound TPO activity, whereas using control IgG the recovery in the unbound fraction was high. These data provide evidence of antibodies against thyroid peroxidase in the serum of patients with AITD and suggest a close link between microsomal antigen and thyroid peroxidase.

Antibodies↗

[Growth rates of two virulence plasmids carrying Yersinia enterocolitica after contamination of heated milk, raw minced pork and vegetables].

The psychrotrophic organism Y. enterocolitica (Y.e.) is able to grow in foodstuffs thus rendering it dangerous for the consumer. Among others, the preparation of food in the kitchen might be an important way of transmission. Not only contaminated raw meat but aswell latently infected humans and pet animals (cats, dogs) may serve as a reservoir for Y.e. contamination. After artificial contamination of UHT milk and vanilla sauce with subsequent storage at 4 degrees C, two Y.e. strains of serovars 0:3 and 0:9 carrying the virulence plasmid multiplied during the first 6 d (9 d) at a rate of 1 log/d (Fig. 1). In pasteurized milk the multiplication rate was 0,65 log/d (Fig.2). Maximum bacterial counts in UHT milk after 15 d were in the order of 10(9) CFU/ml (Fig.4). In pasteurized milk, even minimal inoculation doses of less than 5 CFU/100ml (calculated) produced multiplication of Y.e. to 1,7-27 X 10(4) CFU/ml after 7 d (Tab.1). In this case the milk has been contaminated by an appropriately diluted suspension of faeces from infected mice. When selected colonies were tested on MOX-Agar, a loss of the virulence plasmid could not be observed. In minced fresh pork meat with a bacterial count of 4,2 X 10(6) CFU/g--most of them i.e. 2,6 X 10(6) belonging to Pseudomonas sp.--added Y.e. did not multiply, but viable organisms could be recovered after contamination with only 2,9-6,6 X 10(2) CFU Y.e./g and storage at 4 degrees C for 72 h (Tab.2). Similarly, after contamination of quartered paprika husks and halves of carrots, Y.e. strains could be re-isolated after 15 d storage at 4 degrees C or room temperature if the number of bacteria used for inoculation was in the range of 10(6) (Tab.3). Feeding contaminated carrots to NMRI-mice a calculated number of 5 X 10(3) CFU Y.e./mouse were sufficient to cause infection of part of the animals together with shedding of the organisms through the faeces (Tab.4). These results support the assumption that milk and home made milk products like vanilla sauce create a risk for human health when they are stored for a few days after contamination with Y.e. carrying the virulence plasmid. The same may be true for raw minced pork and fresh vegetables but only if the initial contamination was high. This might occur during food processing procedures.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Glucagon gene sequence. Four of six exons encode separate functional domains of rat pre-proglucagon.

Glucagon, a peptide of 29 amino acids that is produced and secreted by the pancreas, is a regulator of carbohydrate and protein metabolism. Recently, we determined the nucleotide sequence of a mRNA of 1300 nucleotides that encodes rat pre-proglucagon, a polyprotein precursor of glucagon. The polyprotein contains the sequences of glucagon and two glucagon-like peptides arranged in tandem and separated by intervening peptides. Now we report the structure of the gene encoding rat pre-proglucagon. The unique transcriptional unit of the gene spans 10 kilobase pairs and consists of six exons and five introns. Four of the six exons encode distinct functional domains of the pre-proglucagon. The signal sequence, glucagon, and two glucagon-like sequences arranged in tandem are each encoded by a separate exon. A promoter sequence TA-TAAA is located 26 base pairs upstream from the mRNA cap site, and two polyadenylation signals AA-TAAA are present in the 3' untranslated region of the encoded mRNA. The 3' flanking region of the gene contains repetitive sequence DNA.

Amino Acid Sequence↗

Gene encoding parathyroid hormone. Nucleotide sequence of the rat gene and deduced amino acid sequence of rat preproparathyroid hormone.

The nucleotide sequence of the rat parathyroid hormone (PTH) gene was established from a 14.5-kilobase pair fragment of rat liver DNA cloned in bacteriophage Charon 4A. The transcriptional unit of the gene of 2.5 kilobase pairs is interrupted by two introns (1600 and 111 base pairs). Blot hybridization of restriction enzyme digests of rat spleen DNA using 32P-labeled fragments of the cloned PTH gene suggests that the gene is unique and present in a single copy in the genome. A promoter sequence (Goldberg-Hogness or TATA box) is situated 28 base pairs upstream from the point of initiation of transcription which was found by S1 nuclease mapping and by oligonucleotide-primed reverse transcription of rat PTH mRNA. The gene is flanked on its 5' side by repetitive DNA and contains a different, more abundant repetitive DNA on its 3' side. The mRNA encoded by the parathyroid hormone gene consists of 800 +/- 50 nucleotides as determined by electrophoresis on agarose gels. The 5' untranslated region of the mRNA contains three AUG triplets. Only one AUG triplet initiates biosynthesis of preproparathyroid hormone (prepro-PTH). The 3' untranslated segment of the mRNA contains two AATAAA sequences characteristic of polyadenylation signals. The mRNA encodes prepro-PTH, a precursor of PTH of 115 amino acids. When the amino acid sequence of the rat precursor is compared with the analagous bovine and human precursors, it becomes evident that the hormone sequences are highly conserved in two regions of known function near the NH2 terminus and in a third region near the carboxyl terminus whose biologic function, if any, has not yet been defined.

Amino Acid Sequence↗

Somatic mutation creates diversity in the major group of mouse immunoglobulin kappa light chains.

Using a cloned cDNA of a mouse immunoglobulin kappa light chain synthesized in a myeloma MOPC321 (V kappa-21 subgroup C) as a probe we could detect 13 germ line V kappa gene segments. 11 of these were isolated. Using a set of overlapping cloned segments, we showed that nine of these germ line V kappa genes are arranged in two linkage clusters and that they all have the same transcriptional orientation (11, 12, 22). These two clusters occupy 90 and 30 kb of chromosomal DNA and contain six and three V kappa's, respectively. We determined the complete nucleotide sequences of five germ line V kappa's and showed that three of them encode the prototype sequence of V kappa-21 subgroups B, C, and E. None of these five germ line V kappa's encodes the variant amino acid sequences of known V kappa-21 subgroups. We thus conclude that, as in the lambda 1 light chains, the variant V regions are encoded by gene segments derived by a few somatic mutations from the corresponding germ line DNA. Such somatic mutations are not restricted to sequences encoding the hypervariable regions: they also occur in sequences encoding framework regions.

Amino Acid Sequence↗

Pre-proglucagon messenger ribonucleic acid: nucleotide and encoded amino acid sequences of the rat pancreatic complementary deoxyribonucleic acid.

Glucagon, a pancreatic peptide hormone of 29 amino acids that regulates carbohydrate, fat, and protein metabolism, is one of a family of structurally similar regulatory peptides which include GH-releasing hormone, vasoactive intestinal peptide, secretin, and gastric inhibitory peptide. The synthesis of glucagon involves its specific proteolytic cleavage from preproglucagon, a large polyprotein precursor. To facilitate analyses of the cellular processing of pre-proglucagon and to begin studies of the regulation of glucagon gene expression in the rat, we have cloned and sequenced two cDNAs derived from rat neonatal pancreas. The cDNAs represent close to the entire transcriptional sequence of the glucagon gene and encode a pre-proglucagon of 180 amino acids. The coding region of pre-proglucagon contains, in addition to the sequence of glucagon, the sequences of two peptide domains that are related in their structures to glucagon. Glucagon and the two glucagon-like peptides are flanked in the precursor by pairs of basic amino acids characteristic of the sites that are cleaved during the posttranslational processing of pro-hormones. Northern blot analyses indicate the presence of a single mRNA of 1400 +/- 100 nucleotides in the rat pancreas, and results of Southern blotting of rat liver genomic DNA are consistent with the existence of a single chromosomal gene. Comparisons of the nucleotide sequence of the rat pre-proglucagon cDNA with those of the two pre-proglucagons of the anglerfish pancreas encoded by two separate genes indicate that the pre-proglucagon genes probably evolved by intragenic duplications of a DNA segment corresponding to the coding sequences of glucagon and the glucagon-like peptides.

Amino Acid Sequence↗

Parathyroid hormone messenger ribonucleic acid: effects of calcium on cellular regulation in vitro.

The effects of changes in the concentration of extracellular calcium the principal regulatory of PTH secretion, on the cellular levels of the mRNA encoding prepro-PTH, a precursor of PTH, were studied in bovine parathyroid slices in vitro using a sensitive radiodensitometric cDNA hybridization assay and mRNA translation in a wheat germ cell-free system. Although lowering the calcium from 5 to 0.5 mM during 5- to 70-h incubations stimulated the secretion of PTH 4- to 8-fold and pro-PTH synthesis 1.4-fold, the cellular levels of total hybridizable PTH mRNA did not change. However, a 2- to 3-fold increase in PTH mRNA, which could be hybridized to oligo-dT-cellulose [poly(A)-rich], was observed in the slices incubated in 5 mM calcium (suppressed slices). The poly(A)-rich PTH mRNA constituted 30% of the total PTH mRNA in suppressed gland slices and 10% of the total PTH mRNA in stimulated gland slices. PTH mRNA did not decay measurably when RNA synthesis in the gland slices was inhibited with actinomycin D and alpha-amanitin for 6 h. Both the stability and the lack of perturbation of the cellular levels of total of total hybridizable PTH mRNA by changes in the concentration of extracellular calcium suggest that the short term regulation of PTH biosynthesis involves posttranscriptional mechanisms.

Animals↗