RNA polymerase II holoenzyme and transcriptional regulation.

RNA polymerase II holoenzymes isolated from yeast and mammalian cells are large, preassembled complexes that contain some or all of the general transcription initiation factors and many other polypeptides. Recent experiments suggest that these holoenzymes may mediate alterations in chromatin structure and play a key role in regulatory mechanisms that influence transcriptional initiation, RNA chain elongation, RNA processing and transcription termination.

Relationships between relative binding affinity and electrophoretic behavior of rabbit antibodies to streptococcal carbohydrates.

After repeated intravenous injections with Group C streptococcal vaccine, most rabbit antisera were shown to contain one or more IgG antibody components, as revealed by microzone electrophoresis. A procedure for the fractionation of multiple IgG antibody components from such streptococcal antisera is described. Separation is achieved on the basis of differences in relative binding affinities of the antibody components to immunoabsorbent columns. The evidence suggests that the electrophoretic mobility, and thus the net charge of an antibody, bears a reciprocal relationship to its binding affinity for the streptococcal Group C antigens. Furthermore, the relative binding affinity affords another means to assess the functional homogeneity of streptococcal antibodies. A possible relationship between light chain variable-region subclasses and binding affinities of streptococcal antibodies is discussed.

The acute myeloid leukemia-associated protein, DEK, forms a splicing-dependent interaction with exon-product complexes.

DEK is an approximately 45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.

Related RNA polymerase-binding regions in human RAP30/74 and Escherichia coli sigma 70.

RAP30/74 is a heteromeric general transcription initiation factor that binds to mammalian RNA polymerase II. The RAP30 subunit contains a region that is similar in amino acid sequence to the RNA polymerase-binding domain of the Escherichia coli transcription initiation factor sigma 70 (sigma 70). Mammalian RNA polymerase II specifically protected a serine residue in the sigma 70-related region of RAP30 from phosphorylation in vitro. In addition, human RAP30/74 bound to Escherichia coli RNA polymerase and was displaced by sigma 70. These results suggest that RAP30 and sigma 70 have functionally related RNA polymerase-binding regions.

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

RNA polymerase-associated transcription factors.

Proteins that bind to RNA polymerase regulate initiation and termination of transcription in bacteria. Recently, such RNA polymerase-associated proteins were also found to be essential for accurate transcription by eukaryotic RNA polymerase II.

Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein.

We have used protein-blotting and protein affinity chromatography to demonstrate that each of the two glutamine-rich activation domains of the human transcription factor Sp1 can bind specifically and directly to the C-terminal evolutionarily conserved domain of the human TATA box-binding protein (TBP). These activation domains of Sp1 also bind directly to Drosophila TBP but bind much less strongly to TBP from the yeast Saccharomyces cerevisiae. The abilities of the Sp1 activation domains to interact directly with the TBPs of various species correlate well with their abilities to activate transcription in extracts derived from the same species. We also show that a glutamine-rich transcriptional activating region of the Drosophila protein Antennapedia binds directly to TBP in a species-specific manner that reflects its ability to activate transcription in vivo. These results support the notion that TBP is a direct and important target of glutamine-rich transcriptional activators.

The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA.

RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.

Characterization of a six-subunit holo-elongator complex required for the regulated expression of a group of genes in Saccharomyces cerevisiae.

The Elongator complex associated with elongating RNA polymerase II in Saccharomyces cerevisiae was originally reported to have three subunits, Elp1, Elp2, and Elp3. Using the tandem affinity purification (TAP) procedure, we have purified a six-subunit yeast Holo-Elongator complex containing three additional polypeptides, which we have named Elp4, Elp5, and Elp6. TAP tapping and subsequent purification of any one of the six subunits result in the isolation of all six components. Purification of Elongator in higher salt concentrations served to demonstrate that the complex could be separated into two subcomplexes: one consisted of Elp1, -2, and -3, and the other consisted of Elp4, -5, and -6. Deletions of the individual genes encoding the new Elongator subunits showed that only the ELP5 gene is essential for growth. Disruption of the two nonessential new Elongator-encoding genes, ELP4 and ELP6, caused the same phenotypes observed with knockouts of the original Elongator-encoding genes. Results of microarray analyses demonstrated that the gene expression profiles of strains containing deletions of genes encoding subunits of either Elongator subcomplex, in which we detected significantly altered mRNA expression levels for 96 genes, are very similar, implying that all the Elongator subunits likely function together to regulate a group of S. cerevisiae genes in vivo.

RAP30/74: a general initiation factor that binds to RNA polymerase II.

We have previously shown by affinity chromatography that RAP30 and RAP74 are the mammalian proteins that have the highest affinity for RNA polymerase II. Here we show that RAP30 binds to RAP74 and that the RAP30-RAP74 complex (RAP30/74) is required for accurate initiation by RNA polymerase II. RAP30/74 is required for accurate transcription from the following promoters: the adenovirus major late promoter, the long terminal repeat of human immunodeficiency virus, P2 of the human c-myc gene, the mouse beta maj-globin promoter (all of which have TATA boxes), and the mouse dihydrofolate reductase promoter (which lacks a TATA box). RAP30/74 is not required for initiation by RNA polymerase III at the adenovirus virus-associated RNA promoters. Therefore, RAP30/74 is a general initiation factor that binds to RNA polymerase II.

Three functional classes of transcriptional activation domain.

We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.

A motif shared by TFIIF and TFIIB mediates their interaction with the RNA polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces cerevisiae.

Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.

Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH.

The transcriptional activator E2F1 regulates the expression of genes at the G1/S boundary. We have characterized interactions of the E2F1 activation domain with two general transcription factors, the TATA-box binding protein (TBP) and TFIIH. Two distinct binding sites on E2F1 were identified for TBP (amino acids 386-417 and 415-437) each of which supported activation in mammalian cells when expressed as a fusion to a heterologous DNA-binding domain. Neither of these minimal activation domains independently bound TFIIH; rather, the TFIIH binding site of E2F1 overlaps both domains. Loss of TFIIH-binding by E2F1 resulted in a 60-65% reduction in transactivation, suggesting that the E2F1/TFIIH interaction is important, but not essential, for transactivation. The retinoblastoma protein (Rb) binds directly to E2F1 and represses E2F1-mediated transactivation. We have demonstrated that recombinant Rb can compete with TBP and the p62 subunit of TFIIH for binding to immobilized E2F1. A tumorigenic form of Rb deficient in repressing E2F1-mediated transactivation is likewise deficient in displacing TBP from E2F1. We propose that competition between Rb and both TBP and TFIIH for binding to E2F1 is a mechanism by which Rb inhibits transactivation by E2F1.

Direct interaction between two Escherichia coli transcription antitermination factors, NusB and ribosomal protein S10.

The Escherichia coli proteins NusB and ribosomal protein S10 are important for transcription antitermination by the bacteriophage lambda N protein. We have used sucrose gradient co-sedimentation and affinity chromatography with immobilized ribosomal protein S10, a glutathione S-transferase-S10 fusion protein, and NusB to show that NusB binds directly and very selectively to S10. The interaction is non-ionic and has an estimated Kd value of 10(-7) M. We hypothesize that NusB binds to N-modified transcription complexes primarily by interacting with S10.

Interactions of an Arg-rich region of transcription elongation protein NusA with NUT RNA: implications for the order of assembly of the lambda N antitermination complex in vivo.

The E. coli NusA transcription elongation protein (NusA(Ec)), identified because of its requirement for transcription antitermination by the N protein, has an Arg-rich S1 RNA-binding domain. A complex of N and NusA with other host factors binding at NUT sites in the RNA renders RNA polymerase termination-resistant. An E. coli haploid for nusA944, having nine different codons replacing four normally found in the Arg-rich region, is defective in support of N action. Another variant, haploid for the nusAR199A allele, with a change in a highly conserved Arg codon in the S1 domain, effectively supports N-mediated antitermination. However, nusAR199A is recessive to nusA944, while nusA(Ec) is dominant to nusA944 for support of N-mediated antitermination, suggesting a competition between NusA944 and NusAR199A during complex formation. Complex formation with the variant NusA proteins was assessed by mobility gel shifts. NusAR199A, unlike NusA(Ec) and NusA944, fails to form a complex with N and NUT RNA. However, while NusAR199A, like wild-type NusA, forms an enlarged complex with NUT RNA, N, RNA polymerase, and other host proteins required for efficient N-mediated antitermination, NusA944 does not form this enlarged complex. Consistent with the in vivo results, NusA944 prevents NusAR199A but not NusA(Ec) from forming the enlarged complex. The simplest conclusion from these dominance studies is that in the formation of the complete active antitermination complex in vivo, NusA and N binding to the newly synthesized NUT RNA precedes addition of the other factors. Alternative less effective routes to the active complex that allows bypass of this preferred pathway may also exist.

Survey of physician practice behaviors related to diabetes mellitus in the U.S. I. Design and methods.

OBJECTIVE: To conduct a survey among a representative sample of primary care physicians in the U.S. to assess practice behaviors, treatment goals, and beliefs related to management of diabetes mellitus and prevention of its complications. RESEARCH DESIGN AND METHODS: A mail survey with telephone follow-up was conducted among 3481 primary care physicians in active practice in the continental U.S. A stratified probability sample was selected using the files of the American Medical Association and American Osteopathic Association. Four specialties were selected to be included in the study: family physician, general practitioner, internist, and pediatrician. Two versions of a questionnaire were constructed: one for pediatricians containing questions about IDDM only and one for the other three specialties containing questions about both IDDM and NIDDM. Physicians who were not actively engaged in practice or did not see patients with diabetes were excluded. RESULTS: Completed questionnaires were received from 1502 of 3481 sampled physicians. Based on various assumptions of eligibility among nonresponders, an overall response rate to the survey was estimated to be between 65.7 and 86.5%. Discrepancies between specialty identifications as noted on the American Medical Association/American Osteopathic Association files and as self-designated were noted. CONCLUSIONS: This report describes the methodology used in the design and conduct of the survey, and data are provided to document the technical success of survey execution. This report provides the methodological basis for a series of separate reports on demographic characteristics of the physicians, their practices and their patients, and on specific attitudes, beliefs, and practice behaviors of primary care physicians in the U.S. with regard to diabetes mellitus.

Breast implants, rheumatoid arthritis, and connective tissue diseases in a clinical practice.

This study was designed to assess the relationship between breast implants and certain rheumatologic diseases (rheumatoid arthritis and diffuse connective tissue diseases). The study base was a rheumatological practice in Atlanta, Georgia that started in 1982 and began computerizing its records in 1985. The computerized records through May 1992 included 4229 women patients, 150 with breast implants and 721 with a diagnosis of rheumatoid arthritis (RA) and/or one of the connective tissue diseases (CTDs). Of the 721 patients who had been diagnosed as having rheumatoid arthritis (RA) and/or one of the connective tissue diseases (CTDs), 392 had rheumatoid arthritis, 344 had connective tissue disease, 15 had both rheumatoid arthritis and a connective tissue disease, and 33 had more than one connective tissue disease. Of the patients with connective tissue disease, 179 had systemic lupus erythematosus, 64 had scleroderma, 49 had Sjögren's syndrome, 36 had dermatomyositis or polymyositis, and 49 had mixed connective tissue disease. Data were analyzed by univariate and multivariate techniques including logistic regression. Significant variables included age at first visit, income strata, and period of first visit. Analyses were performed for each clinical diagnosis, for all connective tissue diseases together (CTDs), and for those with rheumatoid arthritis and/or connective tissue disease (RA/CTD). Analyses were performed on the total data base and on the records of new patients (1986-1992). The adjusted odds ratio for breast implants among women who entered the practice in 1986-1992 and were diagnosed as having rheumatoid arthritis and/or one of the connective tissue diseases (RA/CTDs) was 0.45 (0.22-0.90), for those with rheumatoid arthritis was 0.61 (0.28-1.49), for those with any of these specific diffuse connective tissue diseases was 0.34 (0.11-1.06) compared to those without the disease. For systemic lupus erythematosus, the odds ratio of 0.24 (0.03-1.75) was based on a single case who had the disease 5 yr before the implant. For Sjögren's syndrome, the odds ratio was 1.67 (0.39-7.13) based on two cases, one of whom had the disease 5 yr before the implant. The calculated odds ratios for scleroderma, dermatomyositis/polymyositis, and mixed connective tissue disease were zero since no cases were diagnosed among the patients with breast implants. This study found no evidence that women with breast implants are at an increased risk for having rheumatoid arthritis or other diffuse connective tissue disease.

Incidence of hospitalized injuries among pregnant women in Maryland, 1979-1990.

INTRODUCTION: Injuries are a leading cause of morbidity among pregnant women. We compared injuries among pregnant women and all women of reproductive age in a large defined population. METHODS: Maryland hospital discharge data files from 1979-1990 were used to identify injuries in pregnant women and in all women 15-44 years of age. Injured pregnant women were defined as those hospitalized with concurrent ICD-9-CM discharge codes for injury and pregnancy. RESULTS: The 80,311 hospitalizations with injury of women 15-44 years old included 2,185 hospitalizations of pregnant women. The incidence of hospitalized injury per 100,000 person-years was 460 for pregnant women and 608 for all women 15-44 years old. Median length of stay and cost per hospitalization were 3.0 days and $1,478 for pregnant women and 4.0 days and $1,666 for all women 15-44 years old. Leading causes of hospitalized injury in pregnant women were poisonings (16.9%), fractures (14.7%), sprains (10.9%), and contusions (8.0%). Compared to all women 15-44 years of age, pregnant women had significantly fewer hospitalizations for dislocations, fractures, poisoning, sprains, and intracranial injuries, and more hospitalizations for contusions and internal injuries. Based on limited information about external causes of injury, pregnant women had significantly fewer hospitalizations for poisoning, drowning/suffocation, and suicide attempts than all women 15-44 years of age. CONCLUSIONS: Many hospitalizations of pregnant women are for relatively minor injuries requiring a short duration of stay, possibly to gauge the impact of the injury on the mother and the fetus. Since most pregnant women receive at least some medical care during pregnancy, prenatal visits represent an ideal time to implement strategies to prevent injuries.

Folic acid in neurodevelopment and child psychiatry.

1. Folic acid deficiency has been associated with diverse neuropsychiatric symptoms. 2. This paper discusses the impact of folate on brain development, maturation and function and reviews the role of folate in psychiatric disorders, particularly childhood disorders. 3. A brief case report examines the use of folate in the treatment of attentional problems in a child with fragile X syndrome.