A general method for the identification of regions suitable for site-directed silent mutagenesis.
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Amino Acid Sequence↗
Biomedical subjects
G D Ehrlich
Publications and source records attributed to G D Ehrlich.
SILMUT: a computer program for the identification of regions suitable for silent mutagenesis to introduce restriction enzyme recognition sequences.
We describe a set of IBM-compatible computer programs designed to selectively identify the potential sites for silent mutagenesis within a target DNA sequence. This program is based on a novel strategy of identifying amino acid motifs compatible with each restriction site (BioTechniques 12:382-384, 1991). The programs can be used to identify the suitability for the introduction of any 6-base nucleic acid sequences, such as restriction enzyme sites in cassette mutagenesis strategies. The Table program generates a table of multiple amino acid motifs for each restriction enzyme, obtained by translating each unique recognition sequence in all three reading frames. The Silmut program, which utilizes the features of Table, will further identify the presence of a match between any amino acid motif of each restriction enzyme and the input target sequence. Minor manipulations of the data base files will enable the individual researcher to identify the potential for introduction of any 6-base sequences by silent mutagenesis.
Base Sequence↗
The inability of human immunodeficiency virus to infect chimpanzee monocytes can be overcome by serial viral passage in vivo.
Studies of lentivirus infection in ruminants, nonhuman primates, and humans suggest that virus infection of macrophages plays a central role in the disease process. To investigate whether human immunodeficiency virus type 1 (HIV-1) can infect chimpanzee macrophages, we recovered monocytes from peripheral blood mononuclear cells of HIV-1-negative animals and inoculated these and control human monocytes with a panel of four human-passaged monocytotropic virus strains and one chimpanzee-passaged isolate. HIV-1 infected human monocytes synthesized proviral DNA, viral mRNA, p24 antigen, and progeny virions. In contrast, except for the chimpanzee-passaged HIV-1 isolate, chimpanzee monocytes failed to support HIV-1 replication when cultured under both identical and a variety of other conditions. Proviral DNA was demonstrated only at background levels in these cell cultures by polymerase chain reaction for gag- and env-related sequences. Interestingly, the chimpanzee-passaged HIV-1 isolate did not replicate in human monocytes; viral p24 antigens and progeny virions were not detected. The same monocytotropic panel of HIV-1 strains replicated in both human and chimpanzee CD4+ T lymphoblasts treated with phytohemagglutinin and interleukin-2. The failure of HIV-1 to infect chimpanzee monocytes, which can be overcome by serial in vivo viral passage, occurs through a block early in the viral life cycle.
Animals↗
Multiple sclerosis, retroviruses, and PCR. The HTLV-MS Working Group.
Previously reported serologic and polymerase chain reaction (PCR)-based findings have suggested an association between the human retrovirus, HTLV-I, and multiple sclerosis (MS). Due to the inherent ability of PCR to produce false-positive results, we developed a set of physical and procedural safeguards to minimize the possibility of molecular carryover. These were applied as part of a blinded, large-scale, multipopulation, multiplex PCR-based study designed to examine this issue of association. Our results do not support the hypothesis that HTLV-I, which plays a role in the pathogenesis of an encephalomyeloneuropathy, HTLV-II, or closely related agents are associated with MS. A concomitant review of the current literature supports this view.
Brain↗
Transmission of human T-lymphotropic virus types I and II by blood transfusion. A retrospective study of recipients of blood components (1983 through 1988). The American Red Cross HTLV-I/II Collaborative Study Group.
We studied results of a "lookback" program involving laboratory testing and interviews of 133 recipients of prior donations from blood donors seropositive for human T-lymphotropic virus types I and II (HTLV-I/II) identified at 28 American Red Cross blood centers. The study was designed to explore the natural course of posttransfusion HTLV-I/II infection among individuals who received blood components from donors subsequently identified as being HTLV-I/II seropositive. Seventeen recipients were seropositive, an apparent transmission rate of 12.8%. Red blood cells and platelets were the implicated components, and red blood cells that were less than 6 days old had a transmission efficiency of 80%. Virus typing enabled documentation of primary and secondary transfusion transmission of HTLV-I and HTLV-II, including the direct transmission of HTLV-II by a donor with a history of intravenous drug use. We conclude that transfusion transmission of HTLV-I/II to approximately 700 recipients per year occurred in the United States before routine donor testing began in 1988.
Adolescent↗
Kaposi's sarcoma in human T-cell leukemia virus type I-associated adult T-cell leukemia.
Kaposi's sarcoma (KS) developed in a patient with human T-cell leukemia virus type I (HTLV-I)-associated adult T-cell leukemia who was treated with a short-term course of monoclonal antibody immunotherapy. The presentation was transient and temporally related to the underlying clinical course. The association of KS in an HTLV-I infected, but not human immunodeficiency virus (HIV)-infected, individual should alert investigators to the occurrence of KS in retroviral-associated diseases other than acquired immunodeficiency disease syndrome. Recognition of the similarities and differences between HTLV-I and HIV infections may provide insights concerning the angiopathogenesis of KS.
Adult↗
Detection of anti-HTLV-I Tax antibodies in HTLV-I enzyme-linked immunosorbent assay-negative individuals.
The HTLV-I tax gene protein (Tax) is not packaged within the mature viral particle from which the proteins for the commercially available enzyme-linked immunosorbent assay (ELISA) are derived. Screening of 162 individuals within a cohort of white intravenous (IV) drug abusers, previously identified as having an increased incidence of HTLV-I infection, demonstrated that seven of them had antibodies to the HTLV-I Tax protein but tested negative in HTLV-I ELISAs and Western blots prepared from purified virion proteins. Three out of 35 individuals in other behaviorally defined high-risk groups also displayed this limited pattern of reactivity to HTLV-I proteins. The presence of the anti-HTLV-I p40/Tax antibodies was determined by radioimmunoprecipitation assay (RIPA), which also revealed low levels of anti-env reactivity. The specificity of the anti-p40 reactivity was confirmed on specific Tax ELISAs and Western blots prepared from recombinantly produced Tax. In vitro gene amplification by the polymerase chain reaction (PCR) was used to establish the presence of sequences homologous to HTLV-I proviral DNA in four/four of these HTLV-I ELISA negative, Tax ELISA/Tax western blot/RIPA positive individuals. These data suggest that the true incidence of HTLV-I infection within high-risk cohorts is greater than previously reported.
Animals↗
A polyclonal CD4+ and CD8+ lymphocytosis in a patient doubly infected with HTLV-I and HIV-1: a clinical and molecular analysis.
HTLV-I is associated with adult T-cell leukemia/lymphoma (ATL) characterized by monoclonal expansions of CD4+ T-lymphocytes. In this report we describe a histologically benign, polyclonal HTLV-I infection in a patient exhibiting both an absolute CD4+ and CD8+ lymphocytosis. Three T-cell lines containing integrated HTLV-I proviral copies established from this patient were initially polyclonal, but with time all grew out the same two clones as determined by analysis of their T-cell antigen receptor beta chain gene rearrangements. The patient subsequently developed pulmonary and nasopharyngeal nodules containing HTLV-I infected cells. Restriction analysis of the patient's HTLV-I provirus revealed no differences from prototype HTLV-I and the tax gene was normally expressed in vivo and in vitro. The patient's T-lymphocytosis and HTLV-I+ pulmonary tract nodules were put into a complete clinical remission by treatment with alkylating agents and steroids. Subsequently, the patient developed a severe immunodeficiency state and expired. Retrospective serologic and gene amplification assays for HIV-1 demonstrated that he had been doubly infected from the time of presentation. Postmortem analysis by polymerase chain reaction revealed the presence of both HTLV-I and HIV-1 in lymphatic tissues and the testes; HIV-1 was also detected in brain tissue.
Acquired Immunodeficiency Syndrome↗
Detection of sequences homologous to human retroviral DNA in multiple sclerosis by gene amplification.
Twenty-one patients with multiple sclerosis, chronic progressive type, were examined for DNA sequences homologous to a human retrovirus. Genomic DNA from peripheral blood mononuclear cells was analyzed for the presence of homologous sequences to the human T-cell leukemia/lymphoma virus type I (HTLV-I) long terminal repeat, 3' gag, pol, and env domains by the enzymatic in vitro gene amplification technique, polymerase chain reaction. Positive identification of homologous pol sequences was made in the amplified DNA from six of these patients (29%). Three of these six patients (14%) also tested positive for the env region, but not for the other regions tested. In contrast, none of the samples from 35 normal individuals studied was positive when amplified and tested with the same primers and probes. Comparison of patterns obtained from controls and from patients with adult T-cell leukemia or tropical spastic paraparesis suggests that the DNA sequences identified are exogenous to the human genome and may correspond to a human retroviral species. The data support the detection of a human retroviral agent in some patients with multiple sclerosis.
Antibodies, Viral↗
Prevalence of human T-cell leukemia/lymphoma virus (HTLV) type II infection among high-risk individuals: type-specific identification of HTLVs by polymerase chain reaction.
The extent of human T-cell leukemia/lymphoma virus type II (HTLV-II) infection and its rate of spread have been difficult to determine owing to the serological cross-reactivity between HTLV-I and HTLV-II. The present study overcame this problem by directly detecting type-specific proviral sequences by means of the polymerase chain reaction (PCR) and liquid hybridization. Screening was performed on a cohort of primarily white intravenous drug abusers (IVDAs), and individuals of other behaviorally defined risk groups from the New York City area. Eleven percent (19 of 169) of the individuals in these high-risk groups were determined by PCR to have HTLV-II proviral infections. One of these patients displayed an exfoliative erythrodermatitis. Thirteen of the 19 subjects were positive in an HTLV-II enzyme-linked immunosorbent assay (ELISA). The remaining six individuals, although negative in the HTLV-II ELISA, were confirmed as HTLV-II positive by analyzing their DNA with a second HTLV-II-specific primer detector system. Four additional individuals were reactive in the HTLV-II ELISA but were PCR-negative for HTLV-II. PCR analysis for HTLV-I revealed that all four were positive for that virus. Thirty-seven percent (seven of 19) of the HTLV-II PCR-positive subjects were also PCR-positive for HTLV-I, and 84% (16 of 19) of the HTLV-II positive individuals were infected with human immunodeficiency virus (HIV-1). Six individuals were triply infected with HTLV-I, HTLV-II, and HIV-1.
Enzyme-Linked Immunosorbent Assay↗
Human T-lymphotropic virus type I-associated benign transient immature T-cell lymphocytosis.
We describe a case of human T-lymphotropic virus type I (HTLV-I)-associated transient benign immature T-cell lymphocytosis in a black female patient, which over the course of several months underwent spontaneous complete remission. The patient presented with a white blood cell count of 20,000/microliter and a T4/T8 ratio of 1.7:1. The majority of cells appeared to be lymphoid in origin, and cell marker analyses established that the circulating lymphocytes were predominantly immature T-cells. HTLV-I was detected at this time by a p19 indirect immunofluorescent slide assay. Over a 1-month period of time the patient's clinical status evolved into a mature T-lymphocytosis with a T4/T8 ratio of 4.5:1. HTLV-I was detected by anti-p19 immunofluorescence by cell sorter analyses and by dot-bloc nucleic acid hybridization. Serological testing demonstrated that the patient had anti-HTLV-I antibodies and antimembrane antibodies specific for an HTLV-I producing cell line. In a competitive HTLV-I ELISA assay only HTLV-I proteins could effectively compete out the seroreactivity. The patient also had a high serum level of soluble interleukin-2 (IL-2) receptors, which is associated with HTLV-I infection. This is the first reported case of immature T-lymphocytosis in a patient infected with HTLV-I. The patient's HTLV-I markers disappeared with time, and her lymphocytosis subsequently spontaneously resolved. She remains disease free and virus negative after 2 years of follow-up study.
Antibodies, Viral↗
Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro.
We evaluated various detection methods to identify amplified human retroviral sequences after Thermus aquaticus-directed polymerase chain reaction (PCR). A combination of hybridization formats and direct incorporation assays provided the most information. This multiphasic approach enabled us to detect specific human T cell leukemia virus type I (HTLV-I)-homologous regions in several HTLV-I-seronegative patients with T cell lymphoma, as well as variants of HTLV-I and human immunodeficiency virus type 1 in patients with prototype disease. In all diagnostic assays designed to detect a particular retrovirus, it was necessary to include a hybridization step, because sequences (endogenous or exogenous) homologous to certain primers were present in most human DNA preparations and yielded discrete products, sometimes of the predicted molecular weight, after amplification. These products could be discriminated by hybridization from amplified prototype proviral sequences. The intensity of the signal generated after hybridization was proportional to input target DNA, an observation making it feasible to quantitatively measure the proviral load in a DNA sample.
AIDS-Related Complex↗
HTLV-I-induced lymphoma mimicking Hodgkin's disease. Diagnosis by polymerase chain reaction amplification of specific HTLV-I sequences in tumor DNA.
A patient with a localized HTLV-I-associated lymphoproliferative disease that was misdiagnosed as Hodgkin's disease is presented. The patient's serum was negative for HTLV-I antibodies by enzyme-linked immunosorbent assay (ELISA), Western blot, and radioimmunoprecipitation. Tumor tissue DNA was negative for HTLV-I by Southern blotting but was positive for distinct HTLV-I sequences when subjected to DNA amplification using the polymerase chain reaction. We conclude that the clinical and pathologic diagnosis of HTLV-I-related lymphoma can be difficult and can be confused with Hodgkin's disease. Extremely sensitive molecular biological techniques may be required to establish a diagnosis of HTLV-I-induced lymphoma.
Adult↗
Clinical and molecular parameters of HTLV-I infection.
The elucidation of the spread of HTLV-I through high-risk groups and the finite but real incidence of HTLV-I seropositivity in normal blood donor populations in the United States indicates that blood and blood products should be screened for this infectious agent. Because of the ability of the provirus to exist in a quiescent state and the long lag time between exposure and seroconversion, it may be necessary to screen potential blood donors for integrated sequences by gene amplification methodologies in addition to standard serologic testing to protect the blood supply. The detection of the HTLV-I virus often requires multiple modes of testing, even in ATL patients. We have characterized by gene amplification several HTLV-I positive lymphoma patients who were seronegative. We have also identified by radioimmunoprecipitation assays intravenous drugs abusers who have antibody solely to the nuclear pX gene product and who do not, therefore, test positive in an ELISA assay prepared from purified virion proteins. All HTLV-I positive patients need to be counseled about the biohazard status of their body fluids. The fact tha only 1 to 2 per cent of HTLV-I infected persons have any diagnosable disease, coupled with the knowledge that the mean time for the onset of clinical manifestations is some 20 to 30 years following conversion to seropositivity, indicates that this is not a virulent pathogen or a highly transforming virus. These epidemiologic data support the notion of HTLV-I's role as a mitogen or first lesion in a multistep pathway to malignancy. These data are also consistent with the idea of rare random cis-activation of one of many cellular oncogenes following a fortuitous integration event.
Antibodies, Viral↗
DNA restriction fragment length polymorphisms at either end of the c-raf-1 locus at 3p25.
We report the identification and characterization of two restriction fragment polymorphisms (RFLPs) associated with the human c-raf-1 protooncogene located on chromosome 3 (3p25). EcoRI digestion produces an RFLP derived from the 5'flanking sequences with a heterozygosity of 28%, and TaqI produces an RFLP representing the 3' flanking sequences with a heterozygosity of 25%. The finding of RFLPs associated with a potential oncogene may provide a method of analysis for determining genetic susceptibilities to certain cancers.
Chromosome Mapping↗
Serum interferon concentrations and retroviral serology in Kawasaki syndrome.
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Antibodies, Viral↗
Quantitative assay of human T-cell leukemia/lymphoma virus transformation.
The in vitro transformation of normal T-lymphocytes by human T-cell leukemia/lymphoma virus (HTLV-I) is possible utilizing cocultivation techniques. We now report on a quantitative assay for HTLV-I transformation. Transformed cell lines were produced by cocultivation of either preactivated (phytohemagglutinin and T-cell growth factor) or nonactivated peripheral blood mononuclear cells with an equal number of lethally irradiated HTLV-I-positive donor cells (MT-2). After 14 days in liquid culture, transformed cells were plated in a 2-layer soft agarose system with or without T-cell growth factor (TCGF). Colony formation among 50 normal controls was observed at varying efficiencies with a mean number of 179 colonies (range, 6-599) in the presence of TCGF (up to a 2-log difference). The day 14 T-cell cultures demonstrated relatively low colony-forming efficiencies (less than or equal to 0.1%) and enhanced colony formation in the presence of TCGF. Day 14 after cocultivation was chosen for this assay based on a dose-response relationship between colony formation and the virus-positive donor cell inoculum and the known kinetics of colony growth of normal activated T-cells. An analysis of individual colonies indicated that they were of target cell origin and HTLV-I positive. Recombinant beta-interferon in increasing concentrations caused a decrease in colony formation as measured in this assay. Long-term cell cultures (2-18 months) showed higher colony-forming efficiencies (up to 1.0%) which were not enhanced by TCGF. The ability to quantitatively evaluate transformation via colony counts will provide an opportunity to study differences in transforming efficiencies attributable to varying target cells, donor cells, or blocking factors such as interferons, drugs, or anti-HTLV-I antibodies.
Cell Line↗
Gamma interferon and AIDS.
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Acquired Immunodeficiency Syndrome↗