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PubMed1977

The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin.

A size class of polysomes was isolated from chick embryonic leg skeletal muscle which synthesized almost exclusively a polypeptide chain with a molecular weight identical to the myosin heavy chain. The mRNA purified from these polysomes was shown to synthesize the 200,000 dalton polypeptide in the wheat germ cell-free translation system. At least 90% of the polypeptide had properties similar to the myosin heavy chain. Isoelectric focusing indicated that the myosin heavy chain synthesized in vitro contained two chains in equal amounts, as did purified embryonic leg skeletal muscle myosin. The kinetics of hybridization of the complementary DNA with an excess of the myosin heavy chain mRNA (MHC mRNA) indicated the presence of two different mRNA sequences. Reassociation of the cDNA to an excess of the DNA of the genome suggest that there is little, if any, reiteration of the myosin heavy chain genes.

Animals↗
PubMed1981

Mammals have multiple genes for individual ribosomal proteins.

The reiteration frequency of the genes that encode the structural proteins of the mammalian ribosome was studied with a set of cloned cDNA probes containing several different mouse r-protein mRNA sequences. Results from a reassociation kinetics analysis, Southern blotting experiments and gene cloning studies collectively indicate that each individual r-protein species is represented by multiple genes in mammals. Among the examples studied, the multiplicity of mouse r-protein genes varied from about 7 to 20, a striking contrast to the low copy numbers observed in less evolutionarily advanced eucaryotes. The multiplicity of individual r-protein genes in humans and rodents is similar.

Animals↗
PubMed1986

Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells.

We have isolated the bovine and human genes for Müllerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Müllerian duct during development of the male embryo. The mRNA sequence of bovine MIS, determined from an analysis of cDNA and genomic clones, codes for a protein of 575 amino acids containing a 24 amino acid leader peptide. The human gene has five exons that code for a protein of 560 amino acids. A comparison of the bovine and human MIS proteins reveals a highly conserved C-terminal domain that shows marked homology with human transforming growth factor-beta and the beta chain of porcine inhibin. Animal cells transfected with the human gene secrete biologically active MIS, which causes regression of the rat Müllerian duct in vitro.

Amino Acid Sequence↗
PubMed1988

A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly.

Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.

Binding Sites↗
PubMed1990

Increased expression of mutant forms of p53 oncogene in primary lung cancer.

Primary lung cancer samples of the major histological types were examined for expression of the tumor suppressor gene p53 by immunohistochemistry. Abnormalities in p53 expression were found in 28 of 40 carcinomas, 14 of 17 squamous tumours showing abnormal p53 expression, whereas no expression of p53 was detectable in 7 carcinoid tumours or in 10 normal lung samples. Direct evidence for homozygous expression of mutant p53 mRNA in representative carcinomas was obtained by means of an asymmetric polymerase chain reaction mRNA sequencing strategy, which allowed sequencing without any cloning step. All the mutations were G to T transversions resulting in mis-sense mutations in aminoacids highly conserved in evolution. Mutation of the p53 gene is the most frequently identified genetic change in human lung cancer; these findings suggest that simple immunohistological methods can provide strong evidence of such mutation.

Adenocarcinoma↗
PubMed1991

Diagrammatic representation of the distribution of DNA bases and its applications.

The frequencies of occurrence of the bases A, C, G and T (or U) in a DNA or mRNA sequence are denoted by a, c, g, t (or u), respectively. Since a + c + g + t = 1, the four real numbers are mapped into a point within a regular tetrahedron. The mapping points are then projected to some planes for further study. We have given several examples of application of this technique. As a by-product of an application we have found an empirical formula on the limit distribution of DNA bases for different kind of organisms, i.e. 1/4 less than or equal to a2 + c2 + g2 + t2 less than 1/3.

Adenine↗
PubMed1985

A single VH-gene associated with a variety of D- and J-segments encodes for a large family of ABPC48-related antibodies induced by antiidiotypic immunization.

Two series of monoclonal antibodies have been obtained from BALB/c mice immunized against two antiABPC48 antiidiotypic antibodies. They are divided into two serologically different classes. Class I antibodies bind only the immunizing antibody; class II antibodies display a broad binding capacity to various antiidiotypic antibodies, and some bind levan, as does ABPC48. Northern blot analyses and partial mRNA sequencing show that all class II antibodies express the VH-gene coding for ABPC48 and UPC10 antilevan antibodies associated with a variety of D- and J-segments. The third hypervariable region of the sequenced antibody with antilevan activity is structurally related to that of ABPC48 and UPC10 antibodies but has a different genetic origin. This study indicates that the identification of idiotype-related antibodies arising from antiidiotypic immunization may be misleading, if based on their antigen-binding properties; and it stresses the importance of structural approaches for the analysis of regulatory mechanisms ruling immune responses.

Amino Acids↗
PubMed1986

Oligodeoxynucleotide stability in subcellular extracts and culture media.

Oligodeoxynucleotide degradation was studied in four systems in order to assess the importance of degradation in hybridization arrest experiments dependent on oligodeoxynucleotides complementary to mRNA sequences. Oligodeoxynucleotides were not detectably degraded over 2 h at 37 degrees C in rabbit reticulocyte lysate or Dulbecco's modified essential medium with 5% fetal calf serum, but were degraded over 2 h in HeLa cell postmitochondrial cytoplasmic extract, and were degraded within 15 min in bovine calf serum.

Autoradiography↗
PubMed1992

Assessment of inactivation of hepatitis A vaccine by compound PCR.

Assuring the complete inactivation of hepatitis A virus (HAV) vaccine commonly requires prolonged tissue culture amplification, followed by detection of virus antigen in cell lysates. A reliable, but faster, alternative procedure is highly desirable since it will permit the prescreening of experimental batches of killed HAV, prior to tissue-culture amplification. We established experimental conditions for simultaneous, polymerase chain reaction (PCR)-based amplification of viral and cellular mRNA sequences from infected cell RNA (compound PCR). Under these conditions, the presence of virus-specific amplified sequences, as detected by Southern blot, allows the identification of incompletely inactivated vaccine batches with a threshold practically identical to that of the more time-consuming subculture and ELISA. Compound PCR is, by its nature flexible enough for adaption to different requirements and it should prove useful for rapid prescreening of vaccine batches and pilot studies for improvement of inactivation protocols.

Base Sequence↗
PubMedDate not supplied

Molecular cloning and expression of an immunodominant 53-kDa excretory-secretory antigen from Trichinella spiralis muscle larvae.

A Trichinella spiralis cDNA expression library was constructed in lambda gt11 from muscle larvae mRNA and immunologically screened to identify genes encoding previously described immunodiagnostic excretory-secretory (ES) antigens. Screening the library with T. spiralis infection serum from swine or rabbit antiserum to T. spiralis ES antigen identified one clone, designated TsA-12, that contains a cDNA transcript 539 bp in length and codes for an apparent 123-kDa beta-galactosidase fusion protein that does not cross-react with Trichuris suis or Ascaris suum infection serum. Western blots of T. spiralis extracts and immunoperoxidase staining of tissue sections from muscle larvae using antibodies to purified TsA-12 demonstrate homology between TsA-12 and the 53 kDa diagnostic antigen from ES products (designated Ts.53) and localize the homologous native antigen to the stichocyte cells of the parasite. ELISA tests using TsA-12 as antigen, detected antibodies to T. spiralis in experimentally-infected mice as early as 14 days post-inoculation with maximum antibody titers being reached at 28 days post-inoculation. The TsA-12 dscDNA hybridizes to mRNA sequences expressed in both the muscle larvae and adult stages; however, concomitant expression of the native antigen is not observed within adult ES products. Southern blots of homologous and heterologous genomic DNAs probed with 32P-labeled TsA-12 dscDNA fragments verify TsA-12 as a T. spiralis specific sequence that is present in multiple copies within the parasite genome.

Amino Acid Sequence↗
PubMed1995

Characterization of the gene family encoding a host-protective antigen of the tapeworm Taenia ovis.

Genomic structure has been determined for a gene encoding a host-protective antigen of the parasitic platyhelminth Taenia ovis. An incomplete cDNA, known as 45W, encodes the protective antigen. Southern hybridisation experiments using 45W cDNA as a probe, revealed that the 45W gene was a member of a multigene family. Differential Southern hybridisation and rapid amplification of cDNA end (RACE) experiments were used to characterise the related genes, allowing the full-length coding region of the 45W encoded antigen to be determined. The gene family comprises a minimum of four members per haploid genome with each member showing varying degrees of 5' and 3' homology with respect to the 45W cDNA. A close homologue of the 45W gene, designated 45S, differed from 45W at 11 of 985 nt comprising the full-length mRNA. Sequencing of several independent RACE products for both 45W and 45S identified a cDNA which may be a product of homologous recombination between these genes, suggesting that the two genes may be alleles. Homologous recombination in genes which encode a host protective antigen such as 45W would provide a mechanism by which antigenic variants could arise.

Amino Acid Sequence↗
PubMed1994

The serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense encodes a variant surface glycoprotein-like protein.

In the Trypanosoma brucei species, T. b. rhodesiense and T. b. gambiense represent the human infective host range variants, while T. b. brucei is lysed upon exposure to a cytotoxic factor in normal human serum. T. b. rhodesiense can occur in a serum-resistant and a serum-sensitive form. The resistance towards normal human serum was shown to be a labile character and to be determined by the environment in which the parasites live. We have clearly demonstrated the presence of RNA transcripts unique to the resistant forms of T. b. rhodesiense. These transcripts encode a protein with VSG characteristics. The DNA fragment isolated previously, which hybridises with the resistance-specific mRNA sequence, appears to be a pseudogene belonging to the same gene family.

Amino Acid Sequence↗
PubMed1994

BON cells display the intestinal pattern of neurotensin/neuromedin N precursor processing.

Antisera towards the bioactive peptides, neurotensin (NT, 13 residues) and neuromedin N (NMN, 6 residues), as well as towards three regions of their 147-residue canine precursor were used to identify and to quantitate precursor-derived peptides in extracts of human BON cells. This cell-line, which was obtained from a human pancreatic carcinoid tumor, constitutively expresses NT/NMN mRNA and secretes NT. Quantitation of seven precursor-derived peptides led us to conclude that BON cells display the intestinal pattern of NT/NMN precursor processing, which is primarily characterized by the production of a large molecular (125 amino acid) form of NMN. Four large molecular components, identified by immunochemical analyses and Western blotting, displayed physico-chemical properties which, for the most part, were consistent with the structures predicted from the partially-known human mRNA sequence. However, as shown previously for these peptides in canine gut, the empirically determined M(r) and pI values were slightly higher than those predicted solely from the amino acid content, perhaps due to the presence of additional substituents. These results suggest that BON cells may provide a good in vitro model in which to study the regulation of intestinal NT/NMN precursor processing and the nature of the enzyme(s) involved.

Amino Acid Sequence↗
PubMed1995

Central administration of antisense oligodeoxynucleotides to neuropeptide Y (NPY) mRNA reveals the critical role of newly synthesized NPY in regulation of LHRH release.

Compelling evidence shows that the episodic and cyclic secretion of hypothalamic luteinizing hormone releasing hormone (LHRH), the primary stimulator of pituitary LH release, is subject to regulation by neuropeptide Y (NPY). We have reported earlier that sequential treatment of ovariectomized (ovx) rats with estrogen and progesterone to stimulate a preovulatory-type LH surge elevated the levels of both NPY and preproNPY mRNA levels in the hypothalamus concomitant with dynamic changes in LHRH activity. The present study was designed to determine whether these elevations in NPY content and gene expression represent new synthesis of NPY that is crucial to elicit LHRH discharge. Ovx, steroid-primed rats received intracerebroventricular injections of an unmodified 20-mer oligodeoxynucleotide (oligo) complementary to the NPY mRNA sequence. Control rats were injected similarly with either saline or the sense or missense oligos. Results showed that control rats displayed a characteristic surge-type elevation in plasma LH levels that was not affected by the administration of missense or sense oligos. However, in rats injected with the antisense oligo, the steroid-induced LH surge was completely blocked. In an additional experiment, NPY peptide levels were measured in microdissected hypothalamic sites following the injection of antisense or missense oligos. NPY antisense oligo administration blocked the significant increases in NPY levels in the median eminence-arcuate area, the medial preoptic area and lateral preoptic area seen in control rats. These results suggest that sequential ovarian steroid treatment augments NPY synthesis in the hypothalamus and this newly synthesized NPY is critical for induction of the LHRH and LH surge.

Animals↗
PubMed1990

Test system for determination of HIV-1 frameshifting efficiency in animal cells.

We have developed a system in animal cells which allows the quantification of frameshifting determined by specific mRNA sequences. The method is based on the expression of an N-terminally extended firefly luciferase gene which requires frameshifting in order to be translated as a functional enzyme. The systems sensitivity is such that it allows the detection of even low efficiency of frameshifting. Our results show that the HIV-1 frameshift sequence including the 3' located stem-loop structure leads to ribosomal frameshifting at a lower level than that described for in vitro systems when tested in several fibroblastoid cell lines.

Amino Acid Sequence↗
PubMed1990

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor.

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.

Apolipoproteins E↗
PubMed1991

Structure and expression of the negative growth factor mouse beta-galactoside binding protein gene.

Following the identification of murine beta-galactoside binding protein (mGBP) as an autocrine negative growth factor we have now isolated and characterized the genomic region spanning the mGBP gene and have determined the 5' end of the transcript by primer extension, S1 mapping and mRNA sequence. The gene is found to be contained within 4 kilobases and composed of four exons of 79, 80, 171 and 197 nucleotides separated by three introns of 1200, 1600 and 193 nucleotides. The DNA region upstream of the 5' end of the transcript contains canonical sequences for eukaryotic promoter elements including CAT and TATA boxes and several DNA motifs for potential transcription regulation. The gene is differentially expressed in a variety of normal tissues.

Amino Acid Sequence↗
PubMed1992

Characterization of transcription and processing from plasmids that use polIII and a yeast tRNA gene as promoter to transcribe promoter-deficient downstream DNA.

Transfer RNA (tRNA) with mutations affecting the internal promoters and thereby causing them to be nontranscribable by polIII (exemplified, e.g., by nematode tDNAPro with large insertions between the two internal promoters) could be transcribed by polIII both in vitro (yeast) and in vivo (oocytes) when cloned behind a yeast tRNAArg gene. PolIII initiated RNA synthesis could also proceed into a downstream structural gene normally read by polIII. the resultant yeast-nematode dimeric tRNA linked in front of mRNA sequences was recognized by processing enzymes to give mature tRNA. Thus, a yeast tRNA gene preceded by its 5' flank can function as a promoter for polIII transcription of any DNA, also of DNA coding for genes that otherwise could not have been expressed either in vitro or in vivo.

Animals↗