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Biomedical subjects

C Morandi

Publications and source records attributed to C Morandi.

At least 37 records · Page 2Linked to original sources
PubMed1990

Search of HIV DNA by polymerase chain reaction in the urine sediments of seropositive individuals.

We have utilized the polymerase chain reaction (PCR) technique to detect proviral sequences of the human immunodeficiency virus (HIV) from urine sediments of HIV seropositive individuals. HIV amplified DNA sequences, easily detectable in peripheral blood cells, were not found in the urine sediments of the seropositive individuals. This finding is in agreement with previous observations that the urines of seropositive individuals are not infective.

DNA, Viral↗
PubMed1989

Secondary structure prediction for RNA binding domain in RNP proteins identifies beta alpha beta as the main structural motif.

In eukaryotic cells transcript processing is strictly dependent upon binding of specific proteins. Nuclear RNA binding proteins share a common domain, which is involved in RNA binding. In order to characterize RNP-RNA interactions we have performed a secondary structure prediction based both on statistical algorithms and comparative analysis of different proteins. A high conservation for secondary structure propensity between different RNPs was observed.

Amino Acid Sequence↗
PubMed1989

Isolation of an active gene encoding human hnRNP protein A1. Evidence for alternative splicing.

Heterogeneous nuclear ribonucleoprotein (hnRNP) core protein A1 is a major component of mammalian hnRNP 40 S particles. We describe the structure of an active A1 gene and report on the partial characterization of the A1 gene family. About 30 A1-specific sequences are present per haploid human genome: 15 such sequences were isolated from a human genomic DNA library. Many corresponded to pseudogenes of the processed type but by applying a selection for actively transcribed regions we isolated an active A1 gene. The gene spans a region of 4.6 x 10(3) base-pairs and it is split into ten exons that encode the 320 amino acid residues of the protein. The amino acid sequence derived from the exon sequences is identical with that deduced from cDNA and reported for the protein. One intron exactly separates the two structural domains that constitute the protein. Each of the two RNA-binding domains in protein A1 is encoded by one exon. Experimental evidence indicates that the A1 gene can encode for more than one protein by alternative splicing. The gene is preceded by a strong promoter that contains at least two CCAAT boxes and two possible Sp1 binding sites, but it lacks a TATA box.

Amino Acid Sequence↗
PubMed1988

cDNA cloning of human hnRNP protein A1 reveals the existence of multiple mRNA isoforms.

Protein A1 is one of the major component of mammalian ribonucleoprotein particles (hnRNP). Human protein A1 cDNA cloning and sequencing revealed the existence of at least two protein isoforms. Among the cDNAs examined, sequence differences were found both in the structural portion, leading to aminoacid changes (Tyr to Phe or Arg to Lys) and in the non translated 3'-region where two T-stretches of different length were observed. Interestingly one of the aminoacid substitutions falls into a consensus sequence common to many RNA binding proteins. Northern blot analysis of poly A+ RNAs from five human tissues revealed two mRNA forms of 1500 and 1900 n due to alternative polyadenylation. Analysis of genomic DNA showed at least 30 A1-specific sequences, some of which correspond to processed pseudogenes. These results suggest that protein A1 is encoded by a multigene family.

Amino Acid Sequence↗
PubMed1986

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single-stranded DNA binding protein (ssDBP) UP I were shown to be cross-reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti-ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re-evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Single stranded DNA binding proteins derive from hnRNP proteins by proteolysis in mammalian cells.

As we have previously demonstrated, mammalian single stranded DNA binding proteins (ssDBP) and heterogeneous nuclear RNA binding proteins (hnRNP proteins) are antigenically and structurally related. In this paper we show that ssDBP are specific proteolytic products of hnRNP core proteins. Proteolysis can be observed in crude extract, both total and nuclear and is not inhibited by the most commonly used protease inhibitors. Such phenomenon can be observed in HeLa cells, human fibroblasts and calf thymus extracts. A trypsin-like protease that cleaves purified hnRNP proteins to give ssDBP of Mr = 24-28 Kd can be purified from HeLa cells. A precursor-product relationship can be established between hnRNP core proteins type A and an ssDBP of 24 Kd (UP1).

Animals↗
PubMed1985

Mammalian single-stranded DNA binding proteins and heterogeneous nuclear RNA proteins have common antigenic determinants.

Antibodies were raised in rabbit against a pure subset of calf thymus single-stranded DNA binding proteins (ssDBPs) and purified by affinity chromatography on antigen-Sepharose. In Western blot experiments these antibodies were shown to react to the same extent with the whole family of bovine ssDBPs, as well as with ssDBPs from HeLa cells. When used to stain total cell extracts from both calf thymus and HeLa cells the antibodies reacted only with bands corresponding to the ssDBPs and with a set of bands of higher molecular weight, whose electrophoretic pattern matched that of the 40S hnRNP core proteins. In effect we observed that purified 40S hnRNP core proteins from HeLa cells were strongly reactive with the antibodies. Moreover after partial tryptic digestion HeLa cells ssDBPs and hnRNPs produced immunoreactive fragments of the same molecular weight and isoelectric point. Extensive structural homologies can thus be evidenced between these two classes of proteins, which share the property of selective binding to single-stranded nucleic acids.

Animals↗
PubMed1984

Microprocessor-based system for spike and eye-movement data acquisition and storage.

A simple system for simultaneous recording of eye position and spike activity during on-line experimentation is described. The system is based on a relatively inexpensive and widely distributed personal computer. While the hardware is configured from commercially available products, the software has been developed in our laboratory. Data acquisition is controlled by a few routines whose logic is outline, together with the main limitations and the possible improvements of the system itself.

Animals↗
PubMed1984

Expression of human dihydrofolate reductase cDNA and its induction by chloramphenicol in Bacillus subtilis.

A bifunctional plasmid (pMP358) able to replicate and to express cloned human dihydrofolate reductase cDNA (cDHFR) in both Escherichia coli and Bacillus subtilis was constructed. The expression of cDHFR in B. subtilis was the result of a deletion that placed the cDNA fragment under the control of the chloramphenicol acetyltransferase (CAT) gene promoter of Staphylococcus aureus plasmid pC194. By sequence analysis of plasmid pMP358, we observed a gene fusion occurring between the cDHFR and the 32nd codon of the CAT gene. We report that such a "hybrid" gene is able to direct the synthesis of a 25-kDal "hybrid" protein, which was found to be inducible by supplementing B. subtilis cells with sublethal doses of chloramphenicol.

Acetyltransferases↗
PubMed1981

Isolation and characterization of dihydrofolic acid reductase from methotrexate-sensitive and -resistant human cell lines.

Dihydrofolic acid reductase has been purified by affinity chromatography to apparent homogeneity from the human HeLa BU-25 cell line and from two methotrexate-resistant variants, one deriving from HeLa BU-25 and the other from the human VA2-B cell line. The purified enzymes from the three sources have been characterized in their physical and enzymatic properties. They were not found to differ significantly as concerns their electrophoretic mobility in polyacrylamide gels under a variety of conditions, their specific dihydrofolic acid reductase and folic acid reductase activities, their Km values for folic acid and TPNH, their sensitivity to methotrexate, and the pH dependence of their folic acid reductase activity. The human dihydrofolic acid reductase has an apparent molecular weight of 21,000 to 22,000, a Km for folic acid of 6.1 to 7.6 X 10(-6) M and a Km for TPNH of 1.6 to 1.7 X 10(-4) M, turnover numbers of about 500 and 65 mol/min/mol of enzyme for the dihydrofolic acid reductase and the folic acid reductase activity, respectively. The values of the above mentioned physical and kinetic parameters are comparable to those reported for the dihydrofolic acid reductase from other animal cell systems. The dihydrofolic acid reductase content of the two-resistant cell lines is at least 200-fold higher than that of the methotrexate-sensitive HeLa BU-25 cell line. The available evidence indicates that this increased dihydrofolic acid reductase content results from a hyperproduction of an enzyme identical or similar to that of the sensitive cells, presumably due to a selective dihydrofolic acid reductase gene amplification, as previously reported for other cell lines of rodent origin.

Drug Resistance↗
PubMed1981

DNA sequence of the araBAD-araC controlling region in Salmonella typhimurium LT2.

The araB and araC genes of Salmonella typhimurium have been cloned onto the plasmid pBR322. Restriction analysis and subcloning of restriction fragments localized these genes to a 4.4 kb DNA fragment. Complementation analysis revealed that the cloned araB and araC genes from S. typhimurium complemented araB and araC mutant strains of escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and araC mutant strains of Escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and ara C mutant strains of S. typhimurium. The DNA sequence was determined for the S. typhimurium araB and araC controlling region and for the initially translated portions of these genes. The nucleotide sequence of the araB promoter was 87% homologous with the same region in E. coli and contained no deletions or insertions relative to the E. coli sequence. The presumed AUG codon corresponding to the amino terminus of the S. typhimurium araC protein was in the same location as in E. coli. There was, however, considerable divergence for the E. coli sequence preceding the translation start site. The nucleotide sequence of the initial 237 bp in the open reading frame of the S. typhimurium araC gene was 78% homologous with the same sequence in E. coli. By comparison, the amino acid sequence for this region was 91% conserved.

Arabinose↗
PubMed1980

Deoxyribonucleic acid sequence of araBAD promoter mutants of Escherichia coli.

The controlling site region for the araBAD operon is defined, in part, by two classes of cis-acting constitutive mutations. The aralc mutations allow low-level constitutive expression of ara-BAD in the absence of the positive regulatory protein coded for by the araC gene, whereas the araXc mutations allow expression of araBAD in the absence of the cyclic adenosine monophosphate receptor protein. Six independently isolated aralc mutations and three independently isolated araXc mutations were cloned onto the plasmid pBR322 using in vitro recombinant deoxyribonucleic acid techniques and in vivo recombination between plasmid and chromosomal deoxyribonucleic acid. The location of these mutations was determined by deoxyribonucleic acid sequence analysis. All of the aralc mutations occurred at position -35 within the araBAD promoter (+1 = messenger ribonucleic acid start for araBAD) and resulted from an AT leads to GC transition. All of the araXc mutations occurred at position -10 within the araBAD promoter and resulted from a GC leads to AT transition. Models are presented to explain the mode of action of the aralc and araXc mutations.

Arabinose↗
PubMed1977

Restoration by T4 ligase of DNA sequences sensitive to "flush" cleaving restriction enzyme.

Fouteen "flush"-ended segments originate from the action of the restriction endonuclease Hae III of Haemophilus aegiptius on the DNA of the colicinogenic factor ColE 1 (A. Oka and M. Takanami, Nature, 264, 191, 1976). They are joined by the T4 polynucleotide ligase. The reaction can be monitored by gel electrophoresis, electron microscopy and resistance to phosphatase of the 5'-32P labelled ends. The joined products are a random recombination of the original segments, and can be cleaved by the same Hae III endonuclease to restore the exact electrophoretic pattern of the Hae III-cut ColE 1 DNA. In a properly diluted mixture of 5'-32P segments treated with T4 ligase, the level of phosphatase resistance is very close to the frequency of circle-formation as determined by electron microscopy: thus, the joining of the "flush"-ends involves the formation of circular structures covalently closed in both strands.

Coliphages↗