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PubMed1991

Cleavage by protease from Staphylococcus aureus V8: an improvement in the sequence analysis of human hemoglobin variants.

Protease from Staphylococcus aureus V8 cleaves either at glutamic residues or at both aspartic and glutamic residues, depending on the experimental conditions. In structural analyses of human hemoglobin variants, the specificity of this enzyme is of considerable interest to localize substitutions occurring in medium or large size peptides as it cleaves in smaller fragments which may be unambiguously characterized. It may also recognize the replacement of an acidic residue by the corresponding amide, or vice versa, avoiding protein sequence analysis. The various aspects of the use of protease V8 are illustrated by the study of four alpha chain hemoglobin variants concerning peptides alpha T-9 and alpha T-12b.

Amino Acid Sequence↗
PubMed1988

Problems in prenatal diagnosis of beta-thalassaemia by fetal blood analysis: beta-chain variant comigrating with gamma chains.

This report describes a couple at risk for beta-thalassaemia in which one spouse was heterozygous for classical high Hb A2 beta-thalassaemia while the other had the compound heterozygous state for beta+-thalassaemia and a beta-chain variant. This variant comigrates on carboxymethyl-cellulose columns (CMC) with gamma-chains, indicating that globin separation on CMC columns could not have been used for fetal diagnosis. The beta-chain variant migrates separately from the other globin chains on HPLC and the respective abnormal haemoglobin can be separated by isoelectrofocusing. Oligonucleotide hybridization showed that both parents were carriers of the beta+ IVS-1, nt 6 mutation. Prenatal diagnosis was successfully accomplished by oligonucleotide analysis on trophoblast DNA. This case indicates that an Antenatal Service should have alternative methods to CMC columns so as to carry out prenatal diagnosis of beta-thalassaemia in uncommon cases.

Blood Protein Electrophoresis↗
PubMed1992

Production and functional analysis of normal and variant recombinant human transthyretin proteins.

The most common form of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy associated with single amino acid changes in the plasma protein, transthyretin. In addition, there are two variants of transthyretin (Ser6 and Thr109) not associated with familial amyloidotic polyneuropathy but with familial euthyroid hyperthyroxinemia, also an autosomal dominant disorder. In these autosomal dominant diseases, most affected individuals are heterozygous and therefore have hybrid forms of the tetrameric plasma transthyretin. In order to study the structure/function relationships of homozygous variant transthyretins, normal human transthyretin and five variant transthyretins (Gly6----Ser, Leu58----His, Thr60----Ala, Ile84----Ser, and Ala109----Thr) were produced in Escherichia coli using the expression vector, pCZ11, and site-directed mutagenesis. These recombinant transthyretin (r-TTR) proteins showed the correct size (14 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis and self-associated into tetramers as determined by size exclusion chromatography. Recombinant normal, Ser6, and Ala60 r-TTRs had an affinity for thyroxine indistinguishable from normal human TTR purified from plasma, whereas His58 and Ser84 r-TTRs had significantly reduced affinity. On the other hand, Thr109 r-TTR had a much higher affinity, probably due to its position within the thyroxine-binding pocket. Expression of mutant transthyretins in E. coli provides the opportunity to study structure/function relationships and amyloid-forming capabilities induced by single amino acid substitutions in the transthyretin molecule.

Amino Acid Sequence↗
PubMed1977

Analysis of thy-1 variants of murine lymphoma cells.

Cells obtained from a radiation-induced T-cell lymphoma of BALB/c mice, RLmale-1, were adapted to long-term tissue culture. Clones of cells were obtained in soft agar and a clonally derived population studied for the frequency of mutation in the expression of the surface antigen Thy-1.2 (OC3H) by immunoselection. The mutation rate was 1.05-1.50 X 10(-6) per cell per generation. Antigenic and structural analysis of prototype positive and negative clones demonstrated clear differences between them.

Animals↗
PubMed1989

Identification of amino acids recognized by syncytium-inhibiting and neutralizing monoclonal antibodies to the human parainfluenza type 3 virus fusion protein.

Neutralizing monoclonal antibodies specific for the fusion (F) glycoprotein of human parainfluenza type 3 virus (PIV3) were used to select neutralization-resistant antigenic variants. Sequence analysis of the F genes of the variants indicated that their resistance to antibody binding, antibody-mediated neutralization or to both was a result of specific amino acid substitutions within the neutralization epitopes of the F1 and F2 subunits. Comparison of the locations of PIV3 neutralization epitopes with those of Newcastle disease and Sendai viruses indicated that the antigenic organization of the fusion proteins of paramyxoviruses is similar. Furthermore, some of the PIV3 epitopes recognized by syncytium-inhibiting monoclonal antibodies are located in an F1 cysteine cluster region which corresponds to an area of the measles virus F protein involved in fusion activity.

Amino Acids↗
PubMed1992

Image processing for cell cycle analysis and discrimination in metastatic variant cell lines of the B16 murine melanoma.

Computer-based image analysis offers a wide range of techniques which can be used to make objective and reproducible detection and measurement of subvisual features of microscopic images of cells. We describe here a prototype imaging system for specimen analysis. Using this system, studies have been carried out on the low metastasis variant F1 and high metastasis variant BL6 of the B16 murine melanoma. Cell cycle analyses have been carried out based on the measurement of integrated nuclear density, nuclear area and nucleocytoplasmic ratio of cells stained using standard procedures. It has been possible to discriminate between the two cell populations on the basis of ploidy. The two cell lines had a similar proportion of cells in the S-phase.

Animals↗
PubMed1989

Hb A2-Wrens or alpha 2 delta 2 98(FG5) Val----Met, an unstable delta chain variant identified by sequence analysis of amplified DNA.

Data are reported for an 85-year-old black make who had an HPFH condition on one chromosome and a suspected 'delta-thalassemia' on the other. Sequence analysis of amplified DNA of an appropriate segment of the delta-globin gene identified a GTG to ATG mutation for codon 98 and thus a Val----Met replacement in the delta chain. This abnormality was confirmed by hybridization of amplified DNA with 32P-labeled synthetic probes and by the amino-acid composition of the isolated tryptic peptide delta T-11. Thus, the 'delta-thalassemia' is caused by the presence of an Hb A2 variant that is considered to be unstable to a similar extent as is Hb Köln, its beta chain counterpart.

Aged↗
PubMed1986

Molecular loss variants of the murine major histocompatibility complex: nonexpression of H-2K antigens associated with marked reduction in H-2K mRNA as determined by oligonucleotide hybridization analysis.

Somatic cell variants of the murine major histocompatibility complex were isolated to study the molecular features required for H-2K gene expression. In vitro selection was performed on a heterozygous [H-2b (Kb,Db) X H-2d (Kd, Dd, Ld)] pre-B lymphoblastoid cell line (R8) for variants that had lost membrane expression of the H-2Kb gene product. Analysis of a number of independently isolated variant cell lines by cytofluorometry with monoclonal antibodies to the Kb, Kd, Db, and Ld antigens revealed a variety of H-2 phenotypes. Variants were classified as either molecular loss for those that had lost K antigen expression only or as haplotype loss for those that no longer expressed the entire H-2b haplotype (i.e., negative for Kb and Db). DNA hybridization analysis with a K gene-specific oligonucleotide indicated that the Kb gene was present in all of the molecular loss variants, suggesting that gene deletion was not responsible for the loss of Kb antigen expression. In contrast, the Kb gene was not detected in haplotype loss variants. For analyzing the mutants at the RNA level, hybridization with H-2-specific synthetic oligonucleotides provided a definitive procedure to identify specific class I gene transcripts in the H-2 heterozygous cell lines. Such analyses were performed on the molecular loss variants and revealed that in a subset of variants (R8.2, R8.96, R8.116, R8.178) there was an absence of Kb mRNA. Kd mRNA was identified for all but one (R8.2) of the Kb mRNA-deficient cell lines, a finding consistent with the serotype assigned to each. Transcription of the linked Db gene was normal. These data demonstrate that a specific alteration for the K gene in several independently selected cell lines gave rise to the altered H-2 phenotype. Such variants offer the potential to analyze the properties of the mammalian genome responsible for controlling expression of individual members of the major histocompatibility complex multigene family.

Animals↗
PubMed1990

Protein analysis of newly isolated variants of echovirus type 18 by electrophoresis and western blotting.

The protein patterns of two newly isolated antigenic variants of echovirus type 18 were compared with those of the prototype strain. Electrophoretic mobilities of VP1 and VP2 of the variants differed from those of the prototype strain. The antiserum against the prototype strain reacted to VP0 and VP2 in the Western blotting experiment. In addition, the variants differed from the prototype strain in growth behavior in the cultured cells.

Blotting, Western↗
PubMed1979

Antigenic drift in type A influenza virus: peptide mapping and antigenic analysis of A/PR/8/34 (HON1) variants selected with monoclonal antibodies.

Variants of A/PR/8/34 (HON1) influenza virus, having hemagglutinin molecules with probably a single altered antigenic determinant, were isolated by growing the virus in the presence of the monoclonal hybridoma antibody PEG-1. The variants were analyzed by peptide mapping and characterized antigenically by using PEG-1 and four other monoclonal hybridoma antibodies to PR8 hemagglutinin. Peptide maps of the large hemagglutinin polypeptide, HA1, from 8 out of 10 variants showed a single changed peptide. This peptide from two of the variants was analyzed, and in each case a serine residue in the wild-type hemagglutinin was replaced by leucine in the variant. Although these eight variants showed identical peptide maps, one could be discriminated antigenically from the others with one of the hybridomas. (The peptide maps represented about one-third of the HA1 molecule.) Of the other two variants, one gave the same HA1 map as the wild type, but could be distinguished antigenically from wild-type virus by two of the hybridomas. The other was unique, and could be distinguished, both antigenically and by peptide mapping, from the other variants. Since a large number of the variants selected with PEG-1 showed the same peptide change, it is likely that this alteration in amino acid sequence (serine to leucine) was responsible for the inability of the variants to bind PEG-1 monoclonal antibody. We do not know, however, whether the changed amino acids were located within the antigenic sites or whether the change occurred somewhere else in the hemagglutinin molecule and altered the determinants through conformational changes.

Amino Acids↗
PubMed1986

Structural analysis of type II variants within the mouse intracisternal A-particle sequence family.

Intracisternal A-particle (IAP) elements are present in multiple copies in the mouse and other rodent genomes. The bulk of this sequence family in Mus musculus consists of 7 Kb long elements, but the majority of IAP sequences involved in known transpositions have been deleted forms. The present study describes a subset of deleted IAP sequences (type II IAP) characterized by insertion of a particular short sequence element (AIIins). AIIins are interspersed and the majority occur as part of the type II IAP elements in the mouse genome. AIIins sequences are absent or in low copy number outside Mus musculus. We have isolated clones containing AIIins from a mouse genomic DNA library and have sequenced three isolates of AIIins and their surrounding IAP sequences to define the detailed structure of type II elements. AIIins are 272, 268 and 264 bp long and 90% homologous in sequence. They are bracketed by 9 bp duplications, suggesting they may be inserted elements. A 75 bp region containing a core enhancer sequence is repeated at the 5' end in type II IAP elements. Insertion into the IAP genome, with potential to encode an integrase function, may have played a role in the amplification of AIIins.

Animals↗
PubMed1990

Prognosis in infiltrating lobular carcinoma. An analysis of "classical" and variant tumors.

A precise histologic and clinical delineation of infiltrating lobular carcinoma, including its variant forms, has been elusive. We studied 230 patients with stage I and II infiltrating lobular carcinoma treated by mastectomy and axillary lymph node dissection. Included were 176 patients with the "classical" or "Indian-file" pattern (IFL) of infiltrating lobular carcinoma, and 54 patients with variant (VAR) histology [solid (SOL), alveolar (ALV), and mixed (MIX) patterns]. IFL patients were younger than VAR patients (52 versus 57 years; p = 0.004), and IFL patients were more likely to be premenopausal (p = 0.013). Microscopic multifocality and intraepithelial ductular extension of LCIS were both more frequent in the IFL group (p = 0.008 and 0.03, respectively). There was no significant difference in tumor size (T1 versus T2), axillary lymph node status, or TNM stage at presentation. Median survival time and time to recurrence was similar in the two groups. Although it was not statistically significant, median survival for stage I and low-axillary lymph node positive stage II IFL patients was better than that of VAR patients. One hundred forty of our 176 IFL patients (80%) could be matched with infiltrating duct carcinoma (IFDC) patients of similar age at diagnosis, menstrual status, tumor size, and axillary lymph node involvement. When stratified by stage, stage I IFL patients had a significantly higher disease-free survival (p = 0.02) than comparable patients with IFDC. There was not a significant difference in disease-free survival when stage II IFL and IFDC patients were compared. The unmodified term "infiltrating lobular carcinoma" should be restricted to lesions exhibiting the classical or Indian-file (IFL) microscopic architectural pattern in approximately 85% of the tumor. Patients with VAR forms of IFL may have a less favorable prognosis and should be distinguished from those with classical IFL for further characterization.

Breast Neoplasms↗
PubMed2019

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

Cell Line↗
PubMed1986

Antigenic variation in Plasmodium chabaudi: analysis of parent and variant populations by cloning.

Nineteen of 22 recrudescent populations of Plasmodium chabaudi AS strain were found to be significantly less sensitive to the protective activity of pools of immune serum, than the parent population from which they were derived. The immune sera were collected from donor mice which had been infected with the parent population and had just reduced the patent primary parasitaemia to subpatent levels. Clones prepared from the parent population (which had previously been cloned) and recrudescent variant populations were tested for their sensitivity to the immune sera. It was found that all the clones from the parent population were sensitive to the immune sera but some were more sensitive than others and that a recrudescent variant population could include both sensitive and insensitive parasites. Two insensitive clones of the recrudescent population were found to be different from each other.

Animals↗
PubMed1979

Proposed replicative role of the NS polypeptide of vesicular stomatitis virus: structural analysis of an electrophoretic variant.

The structural lesion in the temperature-sensitive mutant E1 of the New Jersey serotype of vesicular stomatitis virus has been assigned to the NS protein. Although the packaged wild-type and mutant NS proteins were similarly phosphorylated, the mutant NS protein migrated faster than the wild-type NS protein in polyacrylamide slab gels electrophoresed in the presence of sodium dodecyl sulfate. The resolution appears to be the result of conformational rather than size differences since the two proteins comigrated in polyacrylamide gels which contained 4 M urea in addition to sodium dodecyl sulfate. Peptide maps, obtained by limited proteolysis of 32P-labeled wild-type and mutant NS proteins with Staphylococcus aureus V8 protease and papain, revealed striking differences which suggested that the mutant alteration could involve an aspartic or glutamic acid residue. Since NS proteins obtained from naturally occurring revertants of E1 were indistinguishable from the wild-type protein in all of these analyses, the structural alteration in the mutant NS protein correlates with the functional lesion. Because E1 is defective in the RNA replication pathway at the restrictive temperature, a replicative role is proposed for the NS protein.

Electrophoresis, Polyacrylamide Gel↗