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S Rozenblatt

Publications and source records attributed to S Rozenblatt.

At least 37 records · Page 2Linked to original sources
PubMed1989

DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes.

A number of DNA probes which hybridize to highly abundant DNA sequences of Entamoeba histolytica were developed. Variations in the hybridization patterns of different E. histolytica strains were detected with selected probes. Four types of restriction fragment length patterns were obtained. Of these, the first class belonged to E. invadens and E. histolytica-like var. Laredo. The next two classes consisted of various strains of E. histolytica which were originally isolated from symptomatic patients and possessed pathogenic patterns of isoenzymes (zymodemes), whereas the fourth group contained E. histolytica strains with nonpathogenic zymodemes obtained from asymptomatic carriers. DNA probes, based on DNA sequences specific to E. histolytica isolates with pathogenic and nonpathogenic zymodemes were isolated, and their nucleotide sequences were determined. These probes (P145 and B133) hybridized selectively to DNA of isolates possessing either pathogenic or nonpathogenic isoenzyme patterns. The newly developed probes could be useful for diagnostic purposes and could serve as tools to investigate the molecular basis of pathogenicity and the genetic mechanisms which regulate the variable aggressive behavior of the parasite.

Animals↗
PubMed1988

Nucleotide sequence analysis of an Entamoeba histolytica ferredoxin gene.

A cDNA clone (subclone B) previously isolated from the human parasite Entamoeba histolytica was characterized. DNA sequence analysis of subclone B identified the DNA as that encoding apoferredoxin. E. histolytica ferredoxin cDNA contains unusually short 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. A genomic ferredoxin clone was isolated from E. histolytica DNA, and comparison of genomic and cDNA sequences revealed that the ferredoxin gene is unspliced. The deduced amino acid sequence of E. histolytica ferredoxin resembles clostridial type of ferredoxins, and shows an arrangement of cysteines characteristic for the coordination of 2[4Fe-4S] centres. Of interest is the absence of an aromatic amino acid in the N-terminal region of the protein, a feature which is conserved in clostridial ferredoxins. Southern blot analysis of three different E. histolytica strains (200:NIH, Rahman and HM-1:IMSS) demonstrated the presence of a family of at least two ferredoxin genes. One of these genes is marked by restriction length polymorphisms in different strains of E. histolytica.

Amino Acid Sequence↗
PubMed1987

Entamoeba histolytica: cloning and characterization of actin cDNA.

In order to study gene expression in the human parasite Entamoeba histolytica, a cDNA library of E. histolytica strain 200:NIH was constructed using the phage vector lambda gt10. Three cDNA clones (A, B and C) were selected for further analysis. Each of the three clones hybridized to a distinct mRNA. Two of these mRNAs were translated in vitro after hybrid selection, and yielded distinct translation products. One of these mRNAs, selected by hybridization to clone A, encodes the most abundantly expressed protein in E. histolytica. DNA sequence analysis of this cDNA clone identified the DNA as that encoding actin. The deduced amino acid sequence of E. histolytica actin resembles both cytoplasmic and muscle actins and has an unusual N-terminal glycine residue. We have shown that a family of actin genes is present in E. histolytica. Six different E. histolytica actin clones were obtained from a lambda gt10 genomic library using subcloned cDNA probes. Southern analysis of three different E. histolytica strains (200:NIH, Rhaman, and HM-1:IMSS) revealed at least four different actin genes. Strain HM-1:IMSS, however, differs by the presence of an additional actin gene.

Actins↗
PubMed1986

Accumulated measles virus mutations in a case of subacute sclerosing panencephalitis: interrupted matrix protein reading frame and transcription alteration.

Subacute sclerosing panencephalitis (SSPE) is a fatal disease affecting the human central nervous system several years after acute measles infection. Measles virus (MV) genomes replicating in SSPE brains do not give rise to budding particles and present various defects in gene expression, mostly concerning the matrix (M) protein. For one SSPE case (K), shown previously to be devoid of M protein expression, we examined here in detail the features involved in this defect. In the brain of patient K the normal, monocistronic MV M mRNA was completely substituted by a bicistronic RNA containing the coding sequence of the preceding phosphoprotein (P) gene in addition to the M coding sequence. Analysis of the P-M intercistronic region by direct cDNA sequencing showed that the consensus sequence at this RNA processing site was unaltered but revealed several distant point mutations. cDNA cloning and sequencing of the entire M coding region established that one of the point mutations leads to a stop codon at triplet 12 of the M reading frame. It is unknown whether this defect, explaining by itself the lack of M protein, is related also to the block of M mRNA formation. In addition we note that as much as 1% of the nucleotides differed between two overlapping clones from the same brain. This high sequence variability could possibly account for the diversity of defects observed in MV gene expression in SSPE brains and may be a general phenomenon associated with RNA virus persistence.

Amino Acid Sequence↗
PubMed1986

Subacute sclerosing panencephalitis: detection of measles virus RNA in appendix lymphoid tissue before clinical signs.

An appendix removed 15 days before onset of symptoms of subacute sclerosing panencephalitis was examined retrospectively for measles virus ribonucleic acid (RNA). Tissue sections hybridised in situ to a cloned measles virus probe of deoxyribonucleic acid specific for nucleocapsid protein showed that many cells of the lymphoid tissue contained measles virus RNA. In contrast, only a few infected lymphoid cells were detected in three out of six seropositive controls and none in three seronegative infants. A widespread chronic viral infection of the immune system, established after measles, may promote or even initiate nerve cell infection in subacute sclerosing panencephalitis.

Appendix↗
PubMed1986

Measles virus RNA detected in Paget's disease bone tissue by in situ hybridization.

Morphological and immunocytological studies have demonstrated the presence of paramyxovirus antigens in Paget's bone disease tissue and in particular antigens related to measles virus and respiratory syncytial virus. To examine the relationship between measles virus and Paget's bone disease we used in situ hybridization and a cloned measles virus DNA probe specific for the nucleocapsid protein to detect and locate measles virus RNA sequences in Paget's bone tissue. In five patients with the disease, measles virus RNA sequences were detected not only in 80 to 90% of the multinucleated osteoclasts where there is morphological and immunocytological evidence of measles virus activity but also in 30 to 40% of mononucleated bone cells, mainly osteoblasts, osteocytes, fibroblasts and lympho-monocytes. In contrast, no hybridization was observed in bone tissue from three control patients without signs of Paget's bone disease. These results indicate that the host cell range for measles virus in Paget's disease is more widespread than has been supposed. They also demonstrate the usefulness of the in situ hybridization method to detect viral genetic information in cells where viral antigenic activity is not detectable. These observations further support the hypothesis that measles virus is involved in the pathogenesis of Paget's bone disease.

Bone and Bones↗
PubMed1986

Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences.

The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer.

Amino Acid Sequence↗
PubMed1985

Detection of measles virus RNA in lymphocytes from peripheral-blood and brain perivascular infiltrates of patients with subacute sclerosing panencephalitis.

To clarify the relation between lymphocytes and measles virus in subacute sclerosing panencephalitis, we used in situ hybridization and a cloned measles virus DNA probe, specific for nucleocapsid protein, to detect measles virus RNA sequences in circulating lymphocytes and brain perivascular cuffs of patients with subacute sclerosing panencephalitis. Seventy to 90 per cent of peripheral mononuclear cells from three such patients were found to contain measles virus RNA sequences. In contrast, only a few infected cells were observed in four seropositive adults (0.1 to 5 per cent) and three age-matched children (10 to 15 per cent) used as controls. In one sample of brain tissue from a patient with subacute sclerosing panencephalitis, viral RNA sequences were also detected in nerve cells and in numerous cells from the perivascular infiltrates. In contrast, no hybridization was observed in brain tissue from a patient with herpetic encephalitis and from a patient with postlymphoma encephalitis. We conclude that measles virus has a strong tropism for lymphocytes and nerve cells in subacute sclerosing panencephalitis and that lymphocytes may be involved in the pathogenesis of the disease.

Adult↗
PubMed1985

Sequence homology within the morbilliviruses.

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA extracted from monkey kidney cells infected with canine distemper virus (CDV) was cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing CDV DNA were identified by hybridization to a CDV-specific 32P-labeled cDNA. A cDNA clone containing an insert 1,700 base pairs (CDV 364) has been identified as the reverse transcript of the mRNA coding for the nucleocapsid protein. The size of the mRNA species complementary to this insert is 1,850 nucleotides, as determined by the Northern technique. Hybridization experiments and heteroduplex mapping indicated homology between the central region of the CDV and measles virus nucleocapsid gene. The completion of the nucleotide sequence analysis of the measles virus gene allowed the reconstruction of the entire coding region of the measles virus gene and a comparison with the counterpart sequence of CDV. This comparison delineated three regions: (i) a region of high homology (nucleotides 501 to 1215), in which 77% of the nucleotides and 88% of the encoded amino acids are identical; (ii) a region of moderate homology at the 5' end of the message (nucleotides 1 to 500), in which 59% of the nucleotides and 66% of the encoded amino acids are identical; (iii) a region of little or no homology (nucleotides 1216 to 1625) near the 3' end of the message.

Amino Acid Sequence↗
PubMed1985

Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein.

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1,700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1,500, 1,850, 1,850 and 2,500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

Base Sequence↗
PubMed1985

Measles virus P gene codes for two proteins.

The entirety of the phosphoprotein gene of measles virus has been sequenced. The gene is composed of 1,657 nucleotides and specifies a 507-amino-acid protein (P). A second overlapping reading frame was predicted from the sequence and specifies a 186-amino-acid protein (C). Through the use of antisynthetic peptide antibodies, we show that both proteins are expressed in virally infected cells. Both proteins are expressed from a functionally bicistronic mRNA through independent initiation of ribosomes at the respective AUG codons. Using immunofluorescent microscopy, we localized the C protein in the nucleus and in cytoplasmic inclusions within the infected cells.

Animals↗
PubMed1985

Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus.

A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to immunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA.

Antibodies, Monoclonal↗
PubMed1984

Positive identification of a measles virus cDNA clone encoding a region of the phosphoprotein.

A measles virus-specific cDNA clone, C1-G, that was previously assigned as a hemagglutinin-specific clone has been reassigned as a phosphoprotein-specific clone. The nucleotide sequence of C1-G was used to deduce the amino acid sequence. A synthetic peptide was constructed from a portion of the deduced sequence, and antisera were prepared. The antibodies directed against this synthetic peptide specifically precipitated the phosphoprotein of measles virus and not the hemagglutinin.

Amino Acid Sequence↗
PubMed1983

Localization of measles virus nucleic acid sequences in infected cells by in situ hybridization.

Vero cells were infected with measles virus and hybridized in situ to a cloned DNA fragment containing specific sequences for measles nucleocapsid protein. The DNA was labelled with tritium by nick-translation. The viral RNA were detected in the cytoplasm 21 hrs after infection. In many cells, the probe hybridized to nuclear structures, and in several mitotic cells, to chromosomes. After 36 hrs of infection, hybridization sites were found both in the center and in many nuclei of all the polykaryons. These results indicate that cellular distribution of viral RNA molecules varies in the course of infection. They further suggest that the nucleus plays a more active role than expected in measles virus transcription and replication.

Animals↗
PubMed1982

Cloning and characterization of DNA complementary to the measles virus mRNA encoding hemagglutinin and matrix protein.

Since cloning and characterization of DNA complementary to measles virus mRNA encoding for the nucleocapsid protein (M. Gorecki and S. Rozenblatt, Proc, Natl. Acad, Sci, U.S.A. 77:3686--3690, 1980), two additional measles-specific clones containing different classes of sequences have been characterized. The cloned plasmids contain inserts of 480 and 530 base pairs as shown by agarose gel electrophoresis and electron microscopy. The sizes of the mRNA species complementary to these inserts are 1,700 and 1,550 nucleotides, respectively as determined by the Northern technique. The cloned DNA fragments were further identified as reverse transcripts of the mRNA coding for the glycoprotein and matrix protein of measles virus. The major cell-free translation products of mRNA selected by hybridization to the individual cloned DNAs comigrated with the 70K in vitro products and matrix proteins. One of the cell-free translation products (70K) was also immunoprecipitated specifically with monoclonal antibodies against measles virus glycoprotein.

Base Sequence↗
PubMed1980

Cloning of DNA complementary to the measles virus mRNA encoding nucleocapsid protein.

Double-stranded cDNA synthesized from total poly(A)-containing mRNA, extracted from monkey cells infected with measles virus, has been inserted into Pst cleavage site of Escherichia coli plasmid pBR322 and cloned. A clone containing measles virus DNA sequences was identified by hybridization to a measles virus-specific 32P-labeled cDNA probe prepared from the mRNA of measles virus-infected cells. Cellular sequences in the probe were neutralized by prehybridization with an excess of unlabeled mRNA from uninfected monkey cells. The insert of cloned cDNA isolated contans 1420 base pairs, as shown by agarose gel electrophoresis and electron microscopy. The size of the mRNA complementary to this cloned cDNA is 1750 nucleotides, as determined by the reverse Southern technique. The cloned DNA fragment was further identified as the reverse transcript of the mRNA coding for the nucleocapsid protein of measles virus on the basis that the major cell-free translation product of mRNA selected by hybridization to the cloned DNA comigrated with the nucleocapsid protein and was immunoprecipitated by measles virus-specific antibodies. Subsequently, the cloned DNA was used to detect specific measles virus sequences in the poly(A)-RNA extracted from brain autopsy material from a patient with subacute sclerosing panecephalitis. The cloned DNA can thus be used as a probe to study the structure and expression of the measles genome, and in particular, to study diseases of the central nervous system in which persistent infection with measles virus has been implicated.

Animals↗
PubMed1979

Characterization of measles virus-specific proteins synthesized in vivo and in vitro from acutely and persistently infected cells.

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.

Cell Line↗