Influence of DNA sequence and methylation status on bisulfite conversion of cytosine residues.
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Base Sequence↗
Biomedical subjects
W Schaffner
Publications and source records attributed to W Schaffner.
Complex demethylation patterns at Sp1 binding sites in F9 embryonal carcinoma cells.
The ubiquitous transcription factor Sp1 has been implicated in the mechanism which maintains CpG islands methylation-free. Plasmids containing GC boxes (Sp1 sites) were in vitro methylated at every CpG dinucleotide. After stable introduction into F9 embryonal carcinoma cells, we analysed the methylation of the sequence around the GC boxes with bisulphite sequencing. In agreement with restriction site analysis by other labs, we found preferential demethylation at GC box DNA versus control DNA. However, the bisulphite sequencing which permits the analysis of every CpG site on a given DNA molecule, revealed a complex pattern of methylated and unmethylated sites. Upon prolonged culture the pattern became simpler, with most sites demethylated but certain sites being consistently methylated.
Animals↗
Ehrlichiosis in a golf-oriented retirement community.
BACKGROUND: Ehrlichiosis due to Ehrlichia chaffeensis usually occurs sporadically or in small clusters, with an annual incidence estimated at 3 to 5 cases per 100,000 population in areas of endemic disease. The putative principal vector is the Lone Star tick (Amblyomma americanum). We investigated an outbreak of ehrlichiosis that occurred in June 1993 among members of a golf-oriented retirement community (community A) in Tennessee. The community is densely wooded and borders a wildlife-management area where deer are numerous. METHODS: We conducted a case-control study, using medical-history reviews, serologic testing, and testing with the polymerase chain reaction for E. chaffeensis infection. We also surveyed a sample of 10 percent of the households in community A and in another golf-oriented community (community B) more than 20 miles (32 km) from the wildlife-management area. Survey participants completed a questionnaire and provided specimens for serologic testing. In both communities, searches for ticks were undertaken. RESULTS: Eleven cases of symptomatic ehrlichiosis were identified in the case-control study, 10 of which were in community A (attack rate, 330 per 100,000). Of 311 surveyed residents of community A, 12.5 percent had serologic evidence of past E. chaffeensis infection, as compared with 3.3 percent of 92 in community B (relative risk in community A as compared with community B, 3.9; 95 percent confidence interval, 1.2 to 12.2). The risk of infection was associated with tick bites, exposure to wildlife, golfing, and among golfers, retrieving lost golf balls from the rough. Persons who never used insect repellent were more likely to have had infection than persons who did. In community A, thousands of Lone Star ticks were found; in community B, only three ticks were found. CONCLUSIONS: The high rate of E. chaffeensis infection in community A resulted from its proximity to a wildlife reserve. When outdoor recreational activities are common and concentrations of ticks are high, outbreaks of arthropod-borne zoonoses can be anticipated.
Adolescent↗
Functional domains of the heavy metal-responsive transcription regulator MTF-1.
Metallothioneins (MTs) constitute a class of low molecular weight, cysteine-rich, metal binding proteins which are regulated at the level of gene transcription in response to heavy metals and other adverse treatments. We have previously cloned a zinc finger factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in metallothionein promoters and shown that this factor is essential for basal and heavy metal-induced transcription. Here we report that the C-terminal part of MTF-1 downstream of the DNA binding zinc fingers harbours three different transactivation domains, namely an acidic domain, a proline-rich domain and a domain rich in serine and threonine. When fused to the heterologous DNA binding domain of the yeast factor GAL4 these activation domains function constitutively, i.e. transcription of a GAL4-driven reporter gene is not induced by heavy metals. In search of the region(s) responsible for metal induction, external and internal deletion mutations of mouse and human MTF-1 and chimeric variants thereof were tested with a reporter gene driven by a metal-responsive promoter. The N-terminal part of MTF-1 containing the zinc fingers, which are dependent on zinc for efficient DNA binding, can indeed confer a limited (3- to 4-fold) zinc-responsive transcription when fused to the heterologous activation domain of the viral VP16 protein. Another region containing the acidic and proline-rich activation domains also contributes to metal inducibility, but only in the context of intact MTF-1. This indicates that the activity of MTF-1 results from a complex interplay of different functional domains.
Base Sequence↗
RNA polymerase II C-terminal domain required for enhancer-driven transcription.
The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD may participate in activated transcription through interaction with a high-molecular-weight mediator complex. Such a role would be consistent with observations that some genes are preferentially sensitive to CTD mutations. Here we investigate the function of the mouse RNA polymerase CTD in enhancer-driven transcription. Transcription by alpha-amanitin-resistant CTD-deletion mutants was tested by transient transfection of tissue culture cells in the presence of alpha-amanitin in order to inhibit endogenous RNA polymerase II. Removal of most of the CTD abolishes transcriptional activation by all enhancers tested, whereas transcription from promoters driven by Sp1, a factor that typically activates housekeeping genes from positions proximal to the initiation sites, is not affected. These findings show that the CTD is essential in mediating 'enhancer'-type activation of mammalian transcription.
Amanitins↗
The large subunit of HIV-1 reverse transcriptase interacts with beta-actin.
HIV-1 reverse transcriptase is a dimeric enzyme mainly involved in the replication of the viral genome. A filamentous phage cDNA expression library from human lymphocytes was used to select cellular proteins interacting with HIV-1 reverse transcriptase Affinity selections using the bacterially expressed monomeric large subunit of reverse transcriptase (p66) yielded host beta-actin. This clone was expressed as glutathione-S-transferase fusion protein which was identified by using a specific antibody against beta-actin. Furthermore we show that also the eukaryotic beta-actin binds to either the large subunit of reverse transcriptase or to the Pol precursor polyprotein in vitro. The reverse transcriptase/beta-actin interaction might be important for the secretion of HIV-1 virions.
Actins↗
Positive and negative regulation at the herpes simplex virus ICP4 and ICP0 TAATGARAT motifs.
The control of the ICP0 and ICP4 immediate early genes of herpes simplex virus (HSV) can critically determine the course of viral lytic or latent infections. Their promoters contain so-called TAATGARAT motifs that are activated via a multiprotein complex which includes cellular proteins Oct-1 and HCF and the viral activator (VP16 (= Vmw65, alpha TIF). Relative to the ICP4 promoter TAATGAGAT sequence, the ICP0 promoter motif has a 5' extension that includes a full octamer sequence (ATGCTAATGATAT). It seemed possible that this overlapping octamer site might render the ICP0 promoter element more active by allowing tighter binding of the Oct-1/VP16 complex or more vulnerable to repression by other Oct proteins. Our experiments favor the former possibility. On the one hand, the extended ICP0 site shows stronger binding of the Oct-1/VP16 complex compared to the ICP4 site. Moreover, transcription of a reporter gene with multiple ICP0 sites is strongly activated by VP16 in transfected cells. On the other hand, the ICP0 site is largely refractory toward repression by a different Oct factor (N-Oct2 = Brn1) which competes with Oct-1/VP16 for the site. In marked contrast, multiple copies of the conventional TAATGAGAT motif of ICP4 are poorly activated by VP16, and transcription from this site can be completely repressed by N-Oct2. However, inclusion of the neighboring CGGAAR motifs from the ICP4 promoter, which bind factors GABP alpha and beta, results in a strong synergistic activation. This activity, like that of the complete ICP4 promoter, becomes refractory to repression by competing N-Oct2. Thus the standard TAATGARAT motif of ICP4 is by itself less active and more vulnerable to repression than the extended ICP0 motif, and its activation depends upon synergism with neighboring DNA sites and their cognate factors. This difference between the two types of TAATGARAT motifs may allow for a more complex transcriptional regulation by factor combinations.
Base Sequence↗
A B-cell coactivator of octamer-binding transcription factors.
The octamer motif (ATGCAAAT) paradoxically plays a central role in mediating the activity of both B-cell specific and ubiquitous promoters. It has been widely assumed that the predominantly lymphoid-restricted octamer-binding factor Oct-2 mediates tissue-specific promoter activity, whereas the ubiquitously expressed Oct-1 mediates general promoter activity, but this view has been challenged. Here we use a modified yeast one-hybrid assay to isolate a B-cell factor, Bob1, which associates with either Oct-2 or Oct-1. In transfection experiments, this factor boosts Oct-1-mediated promoter activity and to a lesser extent, that of Oct-2. This coactivation is strictly dependent on the specific interaction with Oct-1 or Oct-2 because deletion of the octamer motif abolishes coactivation. We conclude that Bob1 could represent a new tissue-specific transcriptional coactivator which may convert a ubiquitously expressed transcription factor to a cell-type-specific activator.
Amino Acid Sequence↗
Immunization coverage among infants enrolled in the Tennessee Medicaid program.
OBJECTIVES: To determine immunization coverage of infants receiving Medicaid in Tennessee and to identify risk factors for failure to complete recommended vaccinations by 24 months of age. DESIGN: Retrospective cohort study. SUBJECTS: A total of 33,615 children born in one of three urban Tennessee counties from 1980 through 1989 who were enrolled in Medicaid throughout their first 24 months of life. MAIN OUTCOME MEASURES: Receipt of four diphtheria-tetanus-pertussis, three oral polio, and one measles-mumps-rubella vaccines by 24 months of age (up-to-date), as recorded in computerized county immunization records and Medicaid billing files. RESULTS: Overall, 45% of infants enrolled in Medicaid in the three urban counties completed the recommended vaccinations by 24 months. The proportion of infants up-to-date peaked at 50% for those born in 1982 and 1983, and decreased to 44% for those born in 1989. The only strong independent predictors of immunization completion were number of prior births for the mother, timing of the first immunization, and county of birth. The proportion up-to-date was 56% for first-born children compared with 27% for those whose mothers had at least three prior births; 55% for those whose first immunization was on time compared with 22% for those with a delay in the first immunization; and 63%, 52%, and 37% for infants born in the three respective counties. Maternal age, education, race, and marital status predicted immunization completeness only weakly or not at all. CONCLUSIONS: Of infants born in the three counties in the 1980s who were enrolled in the Tennessee Medicaid program, fewer than half completed their recommended childhood vaccinations by 24 months of age. The large differences in immunization levels between infants enrolled in the Medicaid program in the three counties, not accounted for by differences in demographics, suggest that factors related to the health care and vaccine delivery system have important effects on achieving adequate immunization of these infants.
Child, Preschool↗
Periodicity of eight nucleotides in purine distribution around human genomic CpG dinucleotides.
Mammalian genomes, unlike the genomes of Drosophila and yeast, are characterized by CpG methylation and concomitant CpG depletion, which is caused by the enhanced mutation rate of 5-methylcytosine. To find out whether local nucleotide sequences around existing methylated CpG dinucleotides have common patterns, we analyzed a large population of CpG-poor regions in human DNA, which are typically methylated. We detected a novel periodic variation in the numbers of purine bases around CpGs in the noncoding parts of these sequences. This periodicity of eight nucleotides gradually diminished over 64 nucleotides on each side of the central CpG. Furthermore, the frequencies of the 5' and 3' nearest neighbors of CpGs in CpG-poor regions were biased towards cytosine and guanine, respectively. Such biased sequence contexts may have helped to stabilize CpGs against depletion during mammalian evolution.
Animals↗
Analysis of the heavy metal-responsive transcription factor MTF-1 from human and mouse.
Heavy metal-induced transcription in mammalian cells is conferred by the metal-responsive 70 kDa transcription factor MTF-1 which contains six zinc fingers and at least three activation domains. In previous cell transfection experiments we have shown that the zinc finger region confers an about 3 fold metal inducibility of transcription, due to its differential zinc binding. However, we also noted that human MTF-1 was more metal-responsive than the mouse factor (about 10 fold versus 3 fold, respectively). Here we analyze this difference in more detail by using chimeric human-mouse factors and narrow the critical region to a 64 amino acid stretch immediately downstream of the zinc fingers, overlapping with the acidic activation domain. A short human segment of this region (aa 313-377) confers efficient metal induction to the mouse MTF-1 when replacing the corresponding mouse region. However, high metal inducibility requires an unaltered MTF-1 and is lost when human MTF-1 is fused to the general activation domain of herpesvirus VP16. Wild type and truncation mutants of MTF-1 fused to VP16 yield chimeras of high transcriptional activity, some exceeding the wildtype regulator, but only limited (about 3 fold) heavy metal inducibility.
3T3 Cells↗
Protecting patients when their surgeon or dentist is infected with a blood-borne virus.
The vast majority of surgeons and dentists infected with either hepatitis B or HIV do not transmit these infections to patients. When such transmission from health care provider to patients does occur, the precise mechanism of transmission usually remains enigmatic. Attempts to forestall these rare events have produced substantial debate as well as legislative and regulatory initiatives. The routine immunization of medical and dental students with hepatitis B vaccine will eventually eliminate surgeons and dentists as sources of this infection. Serological screening of surgeons and dentists for HIV infection has been considered impractical. Thus, emphasis has been placed on enhancing measures that reduce the risk of blood contact between health care provider and patient. These include developing safer techniques and procedures. When an infected surgeon or dentist is discovered, a review of their professional circumstances is undertaken. In the US many hepatitis B-infected persons are permitted to continue practice; most HIV-infected persons stop performing invasive procedures. Not completely reassured, some patients inquire of their dentist's or surgeon's HIV antibody status.
Dentists↗
Critical issues surrounding hepatitis B vaccination for adolescents: a roundtable.
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Acute Disease↗
Rhodococcus equi--an increasingly recognized opportunistic pathogen. Report of 12 cases and review of 65 cases in the literature.
Rhodococcus equi, a gram-positive, weakly acid-fast coccobacillus, initially isolated from horses, is becoming increasingly recognized as an important pathogen for immunosuppressed human hosts since the first human case was reported in 1967. A review of the English medical literature yielded 53 cases. During the last 11 years, the microbiology laboratories of the authors isolated the organism from 12 patients. Of the total 65 cases, 60 occurred in immunosuppressed patients with HIV infection, malignant neoplasms, or chronic immunosuppressive therapy. The lung is the most common primary site of infection. Typically, the lesion is densely infiltrated by histiocytes with multiple microabscesses. Intracellular gram-positive coccobacilli are easily demonstrated. R equi grows well on routine non-selective media at 35 degrees C. Previously, many cases may have been missed because the organism resembles oropharyngeal commensal diphtheroids. Clinical information with gram and Kinyoun strains on fresh isolates is helpful in recognizing the possibility of R equi infection.
Acquired Immunodeficiency Syndrome↗
The reporting of communicable diseases: a controlled study of Neisseria meningitidis and Haemophilus influenzae infections.
Surveillance systems for communicable diseases in the United States are primarily passive. We compared the passive reporting system for invasive disease caused by Neisseria meningitidis and Haemophilus influenzae with a concurrent, active laboratory-based system in the four metropolitan counties of Tennessee. The passive reporting system identified approximately 50% of all cases that were identified by the active system and accurately reflected trends in disease occurrence during the study period. Of all reported cases, physicians contributed fewer than 4%. Nearly 40% of all hospitals in the study area did not participate in the passive system. This lack of participation resulted in disproportionately increased reporting of disease among blacks. Inconsistencies in case definition within the state also contributed substantially to underreporting and lack of demographic representativeness of reported cases. The median reporting interval (the time from the onset of disease to transmission of the case report to the Centers for Disease Control and Prevention) was 24 days (range, 5-157 days). Efforts to improve surveillance of those infections for which isolation of a pathogen is tantamount to a diagnosis should concentrate on laboratory-based reporting and the use of currently available computer telecommunication systems.
Centers for Disease Control and Prevention, U.S.↗
Tissue-specific expression of a FMR1/beta-galactosidase fusion gene in transgenic mice.
Fragile X syndrome is one of the most common genetic causes of mental retardation, yet the mechanisms controlling expression of the fragile X mental retardation gene FMR1 are poorly understood. To identify sequences regulating FMR1 transcription, transgenic mouse lines were established using a fusion gene consisting of an E.coli beta-galactosidase reporter gene (lacZ) linked to a 2.8 kb fragment spanning the 5'-region of FMR1. Five transgenic mouse lines showed lacZ expression in brain, in particular in neurons of the hippocampus and the granular layer of the cerebellum. Expression of the reporter gene was also detected in Leydig cells and spermatogonia in the testis, in many epithelia of adult mice, and in the two other steroidogenic cell types, adrenal cortex cells and ovarian follicle cells. Embryonic tissues which showed strong activity of the reporter gene included the telencephalon, the genital ridge, and the notochord. This expression pattern closely resembles the endogenous one, indicating that the 5' FMR1 gene promoter region used in this study contains most cis-acting elements regulating FMR1 transcription.
Animals↗
Coccidioidomycosis among visitors to a Coccidioides immitis-endemic area: an outbreak in a military reserve unit.
An outbreak of coccidioidomycosis occurred in a US Marine reserve unit based in Tennessee after a 3-week training exercise in California that involved substantial exposure to soil and dust. Interviews and serologic testing were done on three occasions (6, 11, and 15 weeks) after the men returned from California, and spherulin skin tests were done at 6 months. Of 27 men, 8 (30%) had evidence of recent coccidioidal infection. Of these, 7 (88%) had an illness consistent with coccidioidomycosis that, despite medical evaluation, was diagnosed incorrectly in 5 men (71%). Diagnosis of coccidioidal pneumonia outside an area in which Coccidioides immitis is endemic is unlikely unless the health care provider is aware that the patient traveled recently. Detection of coccidioidomycosis could be facilitated if organizations that regularly send people to C. immitis-endemic regions were to inform these persons about the risks of infection.
Adult↗
Pathological, physiological, and evolutionary aspects of short unstable DNA repeats in the human genome.
One of the salient features of the mammalian genome is the vast excess of DNA without obvious function, such as repetitive DNAs, spacers, and introns. In recent years, microsatellites, which include short triplet repeats (mostly CAGn and CGGn) and dinucleotide repeats (notably CAn) have gained widespread attention, along with minisatellites which consist of somewhat longer repeat units. Micro- and minisatellites, collectively called variable number tandem repeats (VNTRs), can be highly unstable and display an amazing degree of polymorphism. This property is exploited for gene mapping, for tumor diagnosis, and in forensic medicine. Undue expansion of gene-associated microsatellites is also responsible for some severe genetic diseases, such as fragile X syndrome. Most or all of these diseases are caused by expansion of CAG and CGG triplets. Within protein-coding regions these triplets usually code for polymers of glutamine, serine, alanine or proline. Physiologically, such amino acid repeats are often found in transcription factors and can increase or decrease their activity, depending on the repeat number. Alone or in conjunction with DNA methylation, such repeats may offer a unique opportunity for subtle, semi-stable modulation of gene activity. Also, at least in some plants and perhaps other organisms, a quasi-Lamarckian inheritance is mediated by repetitive DNA. Generally, repetitive DNA sequences, whether represented by short or by long DNA segments, may be beneficial for the evolution of a species.
Base Sequence↗