ABSTRACT
Background: Targeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis. Patients and methods: We developed a comprehensive methylation sequencing assay targeting 9223 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we carried out a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small-cell lung cancer (NSCLC), breast cancer or melanoma and compared results with patients' outcomes. Results: Median methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 versus 85.29; P = 0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 57 (83.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (78.9%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 versus 4.4 months, P < 0.001) and longer time-to-treatment failure (2.8 versus 1.6 months, P = 0.016). Conclusions: Comprehensive targeted methylation sequencing of 9223 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.
Subject(s)
Biomarkers, Tumor/genetics , Cell-Free Nucleic Acids/genetics , DNA Methylation , DNA, Neoplasm/genetics , Neoplasms/classification , Neoplasms/genetics , Adolescent , Adult , Aged , Biomarkers, Tumor/blood , Case-Control Studies , Cell-Free Nucleic Acids/blood , Combined Modality Therapy , DNA, Neoplasm/blood , Female , Follow-Up Studies , Humans , Male , Middle Aged , Neoplasms/blood , Neoplasms/therapy , Prognosis , Survival Rate , Young AdultABSTRACT
Version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for HCV were designed to improve on the performance of first version of the hepatitis C virus (HCV) tests. The new tests were calibrated in international units, the new commonly accepted standard unit of measurement for HCV RNA. The sensitivity of the qualitative tests was enhanced by modifying the specimen processing procedure to achieve a limit of detection 50 IU/ml. The limit of detection for the quantitative tests was 600 IU/ml. Modifications to the amplification reaction mixture and thermal cycling conditions enabled all genotypes to be amplified with similar efficiency. The quantitative tests exhibited a linear range extending from 500 to 500,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 18 to 39%, within the linear range. These data indicate that the version 2. 0 AMPLICOR HCV tests will improve diagnosis of HCV infection and will yield more-accurate titers for prognosis and for monitoring therapeutic efficacy, particularly at low viral loads. Furthermore, it will be possible to compare the performance characteristics and viral load measurements of AMPLICOR tests to those of other tests that adopt the international unit as the standard of measurement.
Subject(s)
Hepacivirus/isolation & purification , Hepatitis C/virology , International System of Units , RNA, Viral/blood , Reverse Transcriptase Polymerase Chain Reaction , Calibration/standards , Genotype , Hepacivirus/genetics , Hepatitis C/diagnosis , Humans , Reagent Kits, Diagnostic , Sensitivity and SpecificityABSTRACT
BACKGROUND/AIMS: Orthotopic liver transplantation has an established role for the treatment of patients with chronic liver failure secondary to hepatitis B virus (HBV) infection. Unfortunately, recurrent infection of the graft can be associated with aggressive disease, and with diminished graft and patient survival. Currently, the role of nucleoside analogues for prevention of graft re-infection is being evaluated. Preliminary results are encouraging, but treatment failure has been associated with emergence of drug-resistant virus. METHODS: We have studied ten consecutive patients who received lamivudine prophylaxis for prevention of HBV graft reinfection. Sequential sera, collected prelamivudine then during treatment before and after liver transplantation, were examined. Conventional serological markers were measured, as were serum viral DNA levels with a sensitive quantitative polymerase chain reaction assay. RESULTS: Lamivudine treatment effected a reduction in serum HBV levels, but six patients still had measurable viral DNA at the time of transplantation. Five patients developed graft re-infection with lamivudine-resistant virus. Resistant virus emerged 8 to 15 months post-transplant. The likelihood of emergence of resistant virus was related to the pre-treatment serum HBV titre. Persistent serum viral DNA positivity and evidence of graft re-infection during the early post-transplant period did not predict the subsequent emergence of resistant virus. CONCLUSIONS: Our observations suggest that the resistant species may be present in the viral quasispecies in the serum and liver of patients with high-level replication prior to lamivudine exposure. The resistant species can persist during lamivudine treatment prior to transplantation, and emerge following transplantation. These observations suggest strategies which might prevent the emergence of drug-resistant species, and imply that graft re-infection may be a preventable phenomenon.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis B virus/isolation & purification , Hepatitis B/surgery , Lamivudine/therapeutic use , Liver Transplantation , DNA, Viral/blood , Hepatitis B/blood , Hepatitis B/drug therapy , Hepatitis B Surface Antigens/blood , Hepatitis B e Antigens/blood , Hepatitis B virus/genetics , Hepatitis B virus/physiology , Humans , Polymerase Chain Reaction , Prognosis , Recurrence , Treatment Failure , Virus ReplicationABSTRACT
Clinical diagnosis of HCV infection is generally accomplished by using immunoserological assays to detect the presence of anti-HCV antibodies. Such immunoserological assays have been approved for blood donor screening, thereby reducing the incidence of post-transfusion hepatitis in the United States. Although useful, immunoserological assays have several limitations. Recent evaluations have shown that interpretation of these immunological tests often is difficult, since 25-90% (depending on the risk group under evaluation) of samples repeatedly reactive in the screening assay are negative on supplemental evaluation with a recombinant immunoblot assay (RIBA) (1,2). Also, the presence of anti-HCV antibodies indicates prior exposure to HCV infection, but cannot be considered a marker for current infection. Nor can anti-HCV antibody levels be used to monitor response to therapeutic agents. Finally, in cases of acute HCV infection resulting from accidental needlestick exposure, many patients fail to produce antibody to HCV (3), which makes diagnosis of HCV infection impossible using immunoserological techniques. At the present time, an immunological assay for direct detection of HCV antigen is unavailable.
ABSTRACT
A significant number of patients with hepatitis C (HCV) treated with interferon (IFN) will initially clear their serum of HCV RNA, but will then have recurrence of viraemia either during or after therapy. One proposed mechanism for relapse is that HCV may persist in peripheral blood mononuclear cells (PBMCs) and that the PBMCs serve as a 'viral reservoir' that is resistant to IFN. To address this hypothesis, we performed serial, quantitative polymerase chain reaction (PCR) of HCV RNA in serum and PBMCs from 26 consecutive patients treated with IFN-alpha2a. Of the 26 patients, 11 (42%) did not clear virus from their serum during therapy and were termed non-responders. Five patients (19%) had sustained clearance of virus from serum and were termed complete responders. The remaining 10 patients (39%) initially eliminated HCV RNA from their serum, but had relapse of viraemia. They were termed partial responders. In all 10 partial responders HCV RNA was undetectable in PBMCs at the same time that it was undetectable in serum. When virus recurred in serum, it was preceded by or occurred at the same time as the return of virus in PBMCs. The results of our study indicate that PBMCs did not serve as an IFN-resistant 'viral reservoir' during therapy. Partial responders who transiently cleared virus from serum also cleared virus from PBMCs and the presence or titre of HCV RNA in PBMCs at the initiation of therapy did not predict response to therapy.
Subject(s)
Antiviral Agents/therapeutic use , Hepacivirus/isolation & purification , Hepatitis C/drug therapy , Interferon-alpha/therapeutic use , Leukocytes, Mononuclear/virology , RNA, Viral/isolation & purification , Adult , Female , Hepacivirus/genetics , Hepacivirus/physiology , Hepatitis C/blood , Hepatitis C/virology , Humans , Interferon alpha-2 , Male , Middle Aged , Polymerase Chain Reaction , Recombinant Proteins , Recurrence , Treatment Outcome , Viremia/diagnosis , Viremia/drug therapy , Viremia/virology , Virus LatencyABSTRACT
BACKGROUND: Several studies from Europe have reported a high prevalence (9% to 32%) of chronic hepatitis C virus (HCV) infection in patients with B-cell non-Hodgkin lymphoma. It has been suggested that HCV plays a role in the pathogenesis of B-cell non-Hodgkin lymphoma. OBJECTIVE: To determine the prevalence of HCV infection in patients with B-cell lymphoma in the United States. DESIGN: Controlled, cross-sectional analysis. SETTING: University medical center. PATIENTS: 120 patients with B-cell lymphoma (78% were Hispanic, 9% were black, 7% were Asian, and 6% were white), 154 patients with other malignant hematologic conditions (control group 1), and 114 patients with nonmalignant conditions (control group 2). MEASUREMENTS: Samples were tested for antibodies to HCV by enzyme-linked immunosorbent assay. Hepatitis C virus RNA was detected by reverse-transcription polymerase chain reaction. Genotyping for HCV was done with genotype-specific primers from the HCV core region. RESULTS: Infection with HCV was detected in 26 patients (22% [95% CI, 15% to 30%]) with B-cell lymphoma compared with 7 of 154 patients (4.5%) in control group 1 and 6 of 114 patients (5%) in control group 2 (P < 0.001). Risk factors for HCV infection were present in 15 patients (60%) with B-cell lymphoma and occurred a median of 15 years before diagnosis of lymphoma. Monocytoid B-cell lymphoma was the most common type of lymphoma found in HCV-positive patients (23% compared with 7% in HCV-negative patients) (P = 0.034). CONCLUSIONS: The prevalence of HCV infection was higher in patients with B-cell non-Hodgkin lymphoma than in controls. The possible role of HCV in the pathogenesis of B-cell lymphoma warrants further investigation.
Subject(s)
Hepatitis C/complications , Lymphoma, B-Cell/complications , Adult , Aged , Aged, 80 and over , California/epidemiology , Cross-Sectional Studies , Female , Hepatitis C/diagnosis , Hepatitis C/epidemiology , Hepatitis C Antibodies/blood , Humans , Male , Middle Aged , Prevalence , RNA, Viral/bloodABSTRACT
Hepatitis G virus (HGV) is a newly described RNA virus that is parenterally transmitted and has been found frequently in patients with chronic hepatitis C infection. To determine the impact of hepatitis G virus co-infection on morbidity and mortality following liver transplantation, we measured HGV RNA by polymerase chain reaction in pre and posttransplantation sera from a cohort of patients transplanted for chronic hepatitis C and a control group of patients transplanted for nonviral causes who were negative for hepatitis C virus (HCV) RNA in serum. The overall prevalence rate of HGV RNA in transplanted patients with chronic hepatitis C was 20.7%. HGV infection was present before transplantation in 13% while it appeared to have been acquired at the time of transplantation in 7.4%. Mean serum alanine aminotransferase activity, hepatic histological activity, and patient and graft survival were similar between HGV-positive and HGV-negative patients. The prevalence rate of HGV RNA in transplanted controls was 64% (P < .01) with a significantly higher rate of acquisition of HGV infection following transplantation (53%, P < .001) when compared with patients with chronic hepatitis C. Mean serum alanine aminotransferase activity was significantly lower in the control patients with HGV infection alone following transplantation than in patients co-infected with hepatitis C (37 +/- 9 vs. 70 +/- 33 U/L, P < .01). Thus, HGV is frequently found in transplantation patients co-infected with hepatitis C although it appears to have minimal clinical impact. In patients transplanted for nonviral causes of end-stage liver disease, a high rate of hepatitis G acquisition at the time of transplantation may occur but does not appear to predispose to chronic hepatitis.
Subject(s)
Flaviviridae/isolation & purification , Hepatitis C/virology , Hepatitis, Viral, Human/virology , Liver Diseases/surgery , Liver Transplantation , Adult , Chronic Disease , Female , Hepatitis C/mortality , Hepatitis, Viral, Human/mortality , Humans , Liver Diseases/mortality , Liver Diseases/virology , Liver Transplantation/adverse effects , Male , Middle Aged , RNA, Viral/blood , Survival AnalysisABSTRACT
Reports suggest that response to interferon-alpha therapy is influenced by both hepatitis C viral genotype and titer. Our aim was to determine if direct, automated, cycle sequencing of the PCR product from an HCV RNA detection assay could be used to reliably determine HCV genotype. In addition, the approach was used to determine the HCV genotype distribution in our patient population and to learn if there was a correlation between HCV genotype and RNA titer that could be used to predict response to treatment. In all 143 consecutive patients were tested for both HCV RNA titer and genotype. Automated, cycle sequencing of PCR product was highly effective and failed to yield a genotype in only 3 (2%) patients. The distribution of HCV genotypes was: 1a (40%), 1b (39%), 2a (2%), 2b (6%), 3a (4%). There were significant differences in the median HCV RNA titers between genotypes 1, 2, and 3. High HCV RNA titers >4.4 x 10(6) copies/ml were only seen in genotype 1. However, the HCV RNA level should not be used as a surrogate marker of genotype because of a significant overlap of titers within the genotypes.
Subject(s)
Hepacivirus/genetics , Hepatitis C/virology , Hepatitis, Chronic/virology , Base Sequence , DNA, Complementary , Female , Genotype , Hepacivirus/classification , Humans , Male , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Viral/blood , RNA, Viral/geneticsABSTRACT
The need to improve the efficacy of anti-viral agents directed against the hepatitis C virus (HCV) has prompted the development of quantitative tests to monitor viraemia levels in relation to therapy. In this respect, we have produced a non-competitive, quantitative PCR assay (Amplicor HCV Monitor) which is based on the single, combined, reverse transcription and amplification of the 5' non-coding region of HCV RNA as well as of an internal standard which serves to assess the overall efficiency of the system. The dynamic range of the assay and its precision allows for an accurate quantification between 1000 and 1,000,000 copies of the viral genome per ml. The results of the first clinical evaluation of the test indicated that determination of viraemia may have predictive value when assessed prior to treatment, as patients with less than 50,000 HCV RNA copies per ml tend to show a long-term response to interferon-alpha (IFN-alpha) treatment. Moreover, a decrease of more than 1.5-2 logarithms occurring at 1 month post-initiation of therapy predicts response in a more timely and accurate fashion than ALT measurement. This may help in a better selection of patients to treat, therapy approaches and schedules and in the optimization of the cost-benefit balance.
Subject(s)
Hepacivirus/genetics , Hepatitis C/virology , Polymerase Chain Reaction/methods , RNA, Viral/blood , Viremia , Chronic Disease , Hepatitis C/blood , HumansABSTRACT
Rational clinical application of quantitative assessments of hepatitis C virus (HCV) RNA depends on an understanding of factors affecting the assay and its intrinsic variability. The effects of three types of blood collection tubes, two storage temperatures, five processing times, and two laboratories on a commercially available quantitative reverse transcriptase PCR assay (AMPLICOR HCV MONITOR) were evaluated. HCV RNA concentrations were assessed in 356 specimens representing 178 aliquots from nine patients. In a multivariate generalized linear model, HCV RNA concentrations decreased when centrifugation was delayed more than 6 h (P = 0.005) and were marginally different between laboratories (P = 0.06), but precentrifugation storage temperature (P = 1.00) and anticoagulation (P = 0.22) had no effect. After adjusting for other factors, the HCV concentration of 95% of a subject's samples were within 0.44 log. Specimens procured for reverse transcriptase PCR-based quantitative HCV testing should be centrifuged within 6 h of collection. Serial assessments should ideally be performed in the same laboratory, and changes in HCV RNA concentration of less than 0.44 log may not be biologically important.
Subject(s)
Hepatitis C/diagnosis , Polymerase Chain Reaction/methods , Adult , Binding, Competitive , Female , Hepatitis C/blood , Humans , Male , Middle Aged , Observer Variation , RNA, Viral/blood , Reagent Kits, Diagnostic , Reproducibility of Results , Sensitivity and Specificity , Specimen Handling , Temperature , Time FactorsABSTRACT
We demonstrated by immunoelectron microscopy that listeriolysin O (LLO), phospholipases and other putative virulence-related proteins produced by Listeria monocytogenes were primarily cell-wall-associated when the bacterium infected Caco-2 tissue culture cell monolayers. Antibodies made to LLO, serogroup 1/2a reacted poorly with serogroup 4b cells and vice-versa, indicating fundamental structural differences in the two proteins. Finally, comet-tail pseudopod structures shown to be involved in cell-to-cell passage of Listeria in Caco-2 cells did not possess detectable Listeria antigens on their anterior surface or within their structure, suggesting that the phagocytic process is primarily host-cell-dependent once it is initiated by the bacterial cell.
Subject(s)
Bacterial Toxins , Heat-Shock Proteins/physiology , Hemolysin Proteins/physiology , Immunohistochemistry/methods , Intestinal Mucosa/microbiology , Listeria monocytogenes/pathogenicity , Antibodies, Bacterial/immunology , Bacterial Proteins/immunology , Bacterial Proteins/physiology , Cells, Cultured , Heat-Shock Proteins/immunology , Hemolysin Proteins/immunology , Humans , In Vitro Techniques , Listeria monocytogenes/immunology , Listeria monocytogenes/metabolism , Listeria monocytogenes/ultrastructure , Microscopy, Electron , Phagocytosis/physiology , Phospholipases/immunology , Phospholipases/physiology , VirulenceABSTRACT
Transcription from the HIV-1 long terminal repeat (LTR) was shown to be inhibited by DNA CpG methylation both in vivo and in vitro. Enzymatic methylation of CpG sites localized within the LTR decreased the transcription of the CAT reporter gene, chloramphenicol acetyltransferase, as assayed by the transient expression of this gene in tissue culture. The inhibitory effect could be initially overcome, in trans, by the transactivator tat. As a function of time, the presence of tat had no observable effect on transcription, within the limits of detection sensitivity, suggesting that the level of basal transcription was reduced to very low levels. This effect is suggestive of the involvement of cellular CpG methylation-dependent inhibitory factors which have been characterized by other laboratories. These data imply that transactivation is reduced to low levels after longer periods of time when the DNA template is sparsely methylated. The transcriptional inhibitory process may involve proteins such as MeCP which may interact with methylated DNA more slowly and/or weakly. Conversely, densely methylated DNA was transcriptionally repressed immediately which suggests the rapid/strong association of the cellular inhibitory factor(s). The transcriptional inhibitory effect was also observed in an in vitro transcription run-off system. These data suggest that the methylation-mediated inhibition of transcription is directly affected by CpG methylation density and may involve other factors.
Subject(s)
DNA, Viral/genetics , Gene Expression Regulation, Viral/genetics , HIV Long Terminal Repeat/genetics , HIV-1/genetics , Nucleotides/genetics , Transcription, Genetic , Cell Line , Cytosine , Electrophoresis, Polyacrylamide Gel , Gene Products, tat/pharmacology , Guanine , HeLa Cells , Humans , Methylation , Plasmids/genetics , T-Lymphocytes/microbiology , Transcriptional Activation , tat Gene Products, Human Immunodeficiency VirusABSTRACT
We describe a spontaneous rough mutant of Listeria monocytogenes that produces reduced amounts of a 60-kilodalton major extracellular polypeptide (p60) as shown by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot analysis. The cells of this mutant are filamentous, do not give rise to smooth wild-type colonies, and produce listeriolysin O in amounts equal to that of the wild-type cells, but they show a reduced virulence in the mouse LD50 model and in the Caco-2 tissue culture virulence assay. Light and electron microscopic studies show that this mutant invades and remains filamentous during in vivo growth in both Caco-2 and 3T6 tissue culture monolayers. The reduced virulence of the rough mutant is not due to the inability of its filamentous forms to invade or to grow in nonprofessional phagocytes since invasion and growth of the smooth wild-type and the rough mutants are comparable in both Caco-2 and 3T6 monolayers.
Subject(s)
Bacterial Proteins/analysis , Listeria monocytogenes/growth & development , Listeria monocytogenes/pathogenicity , Animals , Bacterial Proteins/chemistry , Cell Line , Humans , Listeria monocytogenes/chemistry , Mice , Microbiological Techniques , Microscopy, Electron , Molecular Weight , Mutation , Phenotype , VirulenceABSTRACT
Chromosomal DNA sequences from the 60 kilodalton protein gene of Listeria monocytogenes, amplified by the polymerase chain reaction, were used for restriction fragment length polymorphism differentiation of L. monocytogenes serotypes and other Listeria species. All 24 strains of L. monocytogenes examined produced an extracellular protein of molecular weight 60,000 (p60) as determined by Western blot analysis. Four of six other Listeria species had a protein that cross-reacted to antibodies to p60, but all differed in molecular weight, ranging from approximately 50,000 to 65,000. The gene encoding p60 was amplified from chromosomal DNA in all strains using polymerase chain reaction with a single primer pair. Restriction enzyme digestion with HindIII of the amplified product revealed a restriction pattern that was distinct between serotypes 1/2a and either 4b or 1/2b of L. monocytogenes. Of the other Listeria species, four strains that produced a cross-reacting protein likewise produced a polymerase chain reaction amplification product with the primer pair. Listeria innocua alone had a restriction pattern similar to that of Listeria monocytogenes serotype 4b and 1/2b. Genotypic heterogeneity, as revealed by DNA amplification and restriction endonuclease digestion of the p60 open reading frame, correlates with "electrophoretic type" grouping and may be related to differences in virulence mechanisms of Listeria monocytogenes and other Listeria species.
Subject(s)
Bacterial Proteins/chemistry , Listeria monocytogenes/chemistry , Bacterial Proteins/isolation & purification , Base Sequence , Gene Amplification , Listeria monocytogenes/classification , Listeria monocytogenes/genetics , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
Recently, we reported the partial characterization of bovine lens intrinsic membrane proteins having apparent SDS-PAGE derived molecular mass of 19, 21, and 23 kDa, and determined that they contained identical NH2- terminal amino acid sequences for the first 20 amino acids. From this amino acid sequence information, a mixed synthetic oligonucleotide was constructed and used to screen a calf lens lambda gt11 cDNA library in order to isolate and characterize the cDNA coding for this membrane polypeptide(s). Two separate cDNA clones were isolated and sequenced, and were found to have an identical sequence of 883 bases with an open reading frame coding for a polypeptide of 173 amino acids, having a molecular mass of 19,683 Daltons. The first 20 amino acids of the translated sequence were identical to that determined by our laboratory previously, and the last seven amino acids were identical to that recently determined by another laboratory from analysis of the extracted polypeptides, indicating that this cDNA is the authentic molecule coding for MP19.
Subject(s)
DNA , Eye Proteins/genetics , Membrane Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Cattle , Cloning, Molecular , DNA/isolation & purification , Gene Library , Molecular Sequence Data , Oligonucleotides/chemical synthesis , Open Reading Frames , RNA, Messenger/genetics , RNA, Messenger/isolation & purificationABSTRACT
We have carried out limited microsequence analysis of bovine lens intrinsic membrane proteins having molecular weights of 70, 64, and 38 kD. These three polypeptides all have an identical amino acid terminal sequence, at least for the first 17 amino acid residues, indicating a common origin. When calf lens RNA was hybridized with a labeled antisense oligonucleotide common to the amino acid sequence of these three polypeptides, a single message with an apparent molecular size of 2.6 kb was detected. Together, these results indicate that bovine lens MP70, MP64, and MP38 are products of the same gene and that the lower molecular weight polypeptides are the result of degradation (processing) of lens MP70 at its COOH-terminal end.
Subject(s)
Crystallins/genetics , Eye Proteins/genetics , Membrane Glycoproteins/genetics , Amino Acid Sequence , Animals , Blotting, Northern , Cattle , Connexins , Electrophoresis, Polyacrylamide Gel , Eye Proteins/metabolism , Lens, Crystalline/metabolism , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Oligonucleotide Probes , Protein Processing, Post-Translational , RNA/genetics , Sequence Homology, Nucleic Acid , SheepABSTRACT
We have isolated bovine lens intrinsic membrane proteins (MP) having molecular masses of 19, 21 and 23 kDa. Limited amino acid sequence analysis of the amino-terminal portion of each of these polypeptides revealed a 100% homology in sequence for the number of residues determined (20 amino acids). Northern blot analysis of bovine lens mRNA using a labeled antisense oligonucleotide probe common to the amino acid sequence of these three peptides revealed a single band having an apparent molecular size of 0.8 kb. Taken together, these findings suggest a genetic commonality between these polypeptides.
Subject(s)
Crystallins/analysis , Lens, Crystalline/analysis , Membrane Proteins/analysis , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cattle , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Molecular Weight , RNA/analysisABSTRACT
Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously. We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene. The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located. The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs. The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map. denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it. Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end. Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain. This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence. An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence. Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.