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Biomedical subjects

J DeJong

Publications and source records attributed to J DeJong.

At least 19 recordsLinked to original sources
PubMed2001

TFIIAalpha/beta-like factor is encoded by a germ cell-specific gene whose expression is up-regulated with other general transcription factors during spermatogenesis in the mouse.

TFIIAalpha/beta-like factor (ALF) is a testis-specific counterpart of the large subunit of human general transcription factor TFIIA. Northern analysis shows that ALF mRNA first appears in mouse testis at Postnatal Day 14. Similarly, expression of the general transcription factors TBP, TRF2, TFIIAalpha/beta, TFIIAgamma, and TFIIIB(90) is also increased beginning at Postnatal Day 14, suggesting that there is a coordinated induction of many general transcription factors during male germ cell differentiation. Analysis of male germ cells separated by Staput sedimentation shows that ALF is present in pachytene spermatocytes and haploid spermatids. In addition, in situ hybridization experiments with adult mouse testis shows that ALF is present in haploid spermatids. Searches of the human genome sequence database using the basic local alignment search tool reveal that the ALF and TFIIAalpha/beta(GTF2A1) genes are both composed of nine exons, whereas the TFIIAgamma (GTF2A2) gene is composed of five exons. Furthermore, nucleotide and amino acid comparisons among human and mouse ALF, TFIIAalpha/beta, and TFIIAgamma cDNA sequences show that ALF has diverged more rapidly than either TFIIAalpha/beta or TFIIAgamma. Finally, the ALF and SBLF (Stoned B-Like Factor) sequences present in the chimeric SALF cDNA are both present on human chromosome 2, and an analysis of the corresponding genes suggests a model for the formation of SALF.

Amino Acid Sequence↗
PubMed2000

Functional interactions between an atypical NF-kappaB site from the rat CYP2B1 promoter and the transcriptional repressor RBP-Jkappa/CBF1.

The phenobarbital-inducible rat cytochrome P450 (CYP) 2B1 and 2B2 proteins are encoded by homologous genes whose promoters contain a mammalian-apparent long terminal repeat retrotransposon (MaLR). An NF-kappaB-like site within the MaLR forms multiple protein-DNA complexes with rat liver and HeLa cell nuclear extracts. Using antibody supershift assays, we have identified these complexes as NF-kappaB and RPB-Jkappa/CBF1. Competition assays using a series of single site mutant oligonucleotides reveal that the recognition sites for these two factors overlap. We also show that the CYP2B1/2 NF-kappaB element, but not the Igkappa NF-kappaB element, can repress transcription in vitro when positioned upstream of the heterologous adenovirus major late core promoter. In addition, RBP-Jkappa over-expressed in COS-7 cells repressed expression in vivo from an SV40-luciferase reporter construct that contained the CYP2B1/2 NF-kappaB element. Finally, we observe similar levels of NF-kappaB and RBP-Jkappa binding activities in nuclear extracts prepared from control and phenobarbital-induced rat livers. The results suggest that RBP-Jkappa/CBF1 binds an atypical NF-kappaB site in the CYP2B1/2 promoters and may help to maintain a low level of expression in the absence of inducer.

Animals↗
PubMed2000

The role and limitations of the Cairo International Conference on Population and Development.

Population questions have always aroused controversy, but the International Conference on Population and Development (ICPD) which took place in Cairo in September 1994 was particularly contentious. Yet a consensus emerged among stakeholders previously holding quite divergent positions. A "new paradigm" in population policy emerged from the conference which shifted emphasis from a macro concern with rapid population growth to individual rights in sexuality and reproduction. This consensus has been widely praised, but was far from predictable. It was arrived at through a complicated inter-weaving of interests, movements and intellectual trends, as well as owing much to the particular nature of politics--both global and national--at the time. This paper is an analysis of the policy and substantive significance of the ICPD within the context of the history of UN-sponsored population conferences. It explores how the outcome of the conference was perceived by the various interest groups which played a major role in determining its policy directions, and enumerates some of the critiques of its Programme of Action from different perspectives. It reports on progress and obstacles to implementation of its recommendations within a changed political and economic context than that prevailing in 1994.

Health Policy↗
PubMed1999

Microsomal GST-I: genomic organization, expression, and alternative splicing of the human gene.

In this paper we report the genomic organization of the human microsomal GST-I gene. This gene spans 18 kb, and contains seven exons. Sequences that encode the 155 amino acid open reading frame are present in Exons II, III, IV, the 5'-untranslated region is present in Exons Ia, Ib, Ic, Id, and II, and the 3'-untranslated region is present in Exon IV. Exons Ia, Ib, Ic, Id, and III are alternatively spliced to generate at least six different mGST-I transcripts. The results of EST and PCR analysis show that most mGST-I transcripts terminate within Exon Ib, and primer extension analysis shows these transcripts initiate at three major sites located at 79, 81, and 88 nucleotides upstream of the ATG initiation codon. Sequences surrounding the putative initiation sites are G-C rich, and several Sp1 consensus binding sites were identified. Northern analysis shows that the human GST-I gene is preferentially expressed as a 1.0 kb transcript in liver, and in several other tissues. Finally, a comparison of the mGST-I and PIG12 sequences with those of FLAP, LTC4 synthase, mGST-II, and mGST-III suggests that these proteins are the related products of a dispersed microsomal GST gene superfamily.

Alternative Splicing↗
PubMed1999

Identification of a general transcription factor TFIIAalpha/beta homolog selectively expressed in testis.

In this paper we describe the isolation of a cDNA that encodes a human TFIIAalpha/beta-like factor (ALF). The open reading frame of ALF predicts a protein of 478 amino acids that contains characteristic N- and C-terminal conserved domains separated by an internal nonconserved domain. In addition, a rare ALF-containing cDNA, which possesses an extended N terminus (Stoned B/TFIIAalpha/beta-like factor; SALF) has also been identified. The results of Northern and dot blot analyses show that ALF is expressed almost exclusively in testis; in contrast, TFIIAalpha/beta and TFIIAgamma are enriched in testis but are also widely expressed in other human tissues. Recombinant ALF (69 kDa) and TFIIAgamma (12 kDa) polypeptides produced in Escherichia coli form an ALF/gamma complex that can stabilize TBP-TATA interactions in an electrophoretic mobility shift assay. The ALF/gamma complex is also able to restore transcription from the adenovirus major late promoter using HeLa cell nuclear extracts that have been depleted of TFIIA. Overall, the data show that ALF is a functional homolog of human general transcription factor TFIIAalpha/beta that may be uniquely important to testis biology.

Adenoviridae↗
PubMed1996

Interaction of the human T-cell lymphotropic virus type 1 tax transactivator with transcription factor IIA.

The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation. Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein, NF-kappaB, and serum response factor. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. On the basis of several independent assays, we now report a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35-kDa (alpha) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized mutants with point mutations in Tax, M32 (Y196A, K197S) and M41 (H287A, P288S), which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay. Second, a glutathione-S-transferase (GST) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes, Tax and TFIIA were associated. Finally, TFIIA facilitates Tax transactivation in vitro and in vivo. In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA. In addition, transfection of human T lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation.

Amino Acid Sequence↗
PubMed1995

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Animals↗
PubMed1995

Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription.

The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts.

Amino Acid Sequence↗
PubMed1994

Characterization of the highly conserved TFIIA small subunit from Drosophila melanogaster.

TFIIA is a general transcription factor that modulates class II transcription initiation in vitro by functionally and physically interacting with TFIID or the derived TATA-binding protein (TBP). TFIIA was previously purified from human, bovine, rat, and yeast sources and has recently been identified in association with TFIID in Drosophila. Here, we report the cloning of a cDNA encoding the 12.5-kDa subunit of TFIIA from Drosophila melanogaster (dTFIIA-S) and the identification of a partial dTFIIA-S gene in Drosophila virilis. The deduced amino acid sequence of dTFIIA-S indicates a high degree of homology to the small TFIIA subunit from yeast and to a partial TFIIA-S cDNA identified in rice. A hybrid TFIIA consisting of recombinant dTFIIA-S and the recombinant human TFIIA/alpha gene product mimics natural TFIIA activity in a TBP-dependent DNA binding assay. The promoter complex formed with this hybrid TFIIA depends upon the TATA element and can be efficiently incorporated into a higher order preinitiation complex upon addition of TFIIB. The presence of dTFIIA-S within the TBP-TFIIA-promoter complex was demonstrated using anti-dTFIIA-S antiserum. Finally, the ability of recombinant dTFIIA-S to reconstitute transcriptionally active TFIIA was demonstrated in a well defined human transcription system.

Amino Acid Sequence↗
PubMed1993

A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA.

TFIIA is a transcription factor that, by interacting with the TATA-binding subunit (TBP) of TFIID, modulates transcription initiation by RNA polymerase II in vitro. By use of a mobility shift assay, TFIIA was purified from HeLa cells as a complex of 35-, 19-, and 12-kD subunits. Oligonucleotides were used to isolate a human cDNA clone, hTFIIA/alpha, which encodes a 55-kD protein with homology to the product of the yeast gene TOA1. The open reading frame of hTFIIA/alpha contains peptide sequences obtained from both the p35 and p19 subunits of natural human TFIIA, and thus encodes these two subunits. Consistent with this, antiserum raised against the 55-kD hTFIIA/alpha-encoded protein reacted with both the p35 and p19 subunits of natural TFIIA, and the recombinant protein could functionally replace those subunits in a mobility shift assay with renatured p12. An efficient affinity purification for natural human TFIIA was suggested by the sequence of the hTFIIA/alpha protein and demonstrated biochemically. Finally, transcription from the adenovirus major late promoter was greatly reduced in nuclear extracts depleted with anti-TFIIA/alpha serum and was restored to original levels by the readdition of purified human TFIIA.

Amino Acid Sequence↗
PubMed1993

Substance abuse among subjects screened out from an alcoholism research program.

Three hundred and eight subjects were screened over the phone for admission to an inpatient alcohol treatment research unit. Using a structured interview, the prospective patients were asked questions regarding demographics, drinking history, previous treatments, physical health, family history, and a detailed history of past and present substance use. Drug use was studied as regular use versus no use or brief experimental use of five drug categories: cannabinoids, stimulants, sedatives, opiates, and hallucinogens. Fifty-one percent of the men and 48% of the women reported regular use of one or more of the drugs in addition to alcohol. For women, the amount of alcohol intake was positively correlated with use of stimulants (r = .32, p = .001), cannabinoids (r = .24, p = .019), sedatives (r = .30, p = .003), and hallucinogens (r = .30, p = .003). For men, correlations between the amount of alcohol intake and drug use were weaker but significant for stimulants (r = .21, p = .002), opiates (r = .15, p = .028), and hallucinogens (r = .15, p = .029). Women with alcoholic mothers displayed higher alcohol intake than women with nonalcoholic mothers (p = .02) and also showed more frequent use of most drugs. Although men with alcoholic fathers also showed greater alcohol intake compared to men with nonalcoholic fathers, the two groups did not differ in drug use. Younger subjects of both sexes were more likely to use cannabinoids, stimulants, opiates, and hallucinogens. Alcohol and sedative use was relatively constant across all age groups.

Adult↗
PubMed1992

Factors associated with recidivism in a criminal population.

The present study is a follow-up of a sample of 348 men convicted of manslaughter, attempted manslaughter, or arson who were released from incarceration. Multiple factors assessed at the time of incarceration, including demographic, behavioral, family history, and biochemical variables, and psychiatric diagnoses were used in an attempt to discriminate between those who became recidivists during the follow-up period and those who did not. Violent recidivism was most strongly associated (sensitivity of 90%) with impulsivity of the original crime in killers and attempted killers; for arsonists, having made a suicide attempt was the strongest predictor (68% sensitivity). For predictive purposes, both single factor associations and multiple entries into discriminant analysis produced too many false-positives, i.e., the high rate of false designation as recidivist remained a problem.

Adolescent↗
PubMed1992

TFIIA induces conformational changes in TFIID via interactions with the basic repeat.

DNA-binding studies with Saccharomyces cerevisiae TFIID point mutants indicated that TFIIA interacts with the basic repeat region of TFIID and induces structural changes. The latter was shown by the ability of TFIIA to compensate for TFIID point mutants defective for DNA binding. Interaction with TFIIA also rendered TFIID binding temperature independent, thus mimicking the effect of removing the nonconserved N terminus of TFIID. In addition, N-terminal truncation of the TFIID point mutants defective for DNA binding mimicked the ability of TFIIA to restore DNA binding of those mutants. Taken together, these results suggest that TFIIA enhances TFIID binding to DNA by eliminating an otherwise inhibitory effect of the nonconserved N terminus of TFIID. Furthermore, analyses of TFIID contact points on DNA and binding studies with TATA-containing oligonucleotide probes showed that TFIIA decreases the effect of sequences flanking the adenovirus major late TATA element on TFIID binding to DNA, suggesting a possible role of TFIIA in allowing TFIID to recognize a wider variety of promoters.

Base Sequence↗
PubMed1991

Mental disorders among alcoholics. Relationship to age of onset and cerebrospinal fluid neuropeptides.

Eighty-one percent of 339 alcoholics participating in a research program were found to have associated mental disorders. Alcoholics with onset of heavy drinking before 20 years of age had significantly more antisocial personality traits, drug abuse, bipolar disorder, panic disorder, suicide attempts, and paternal alcoholism than alcoholics with onset after age 20 years. Alcoholics with onset before and after 20 years of age also differed significantly from each other for cerebrospinal fluid concentrations of diazepam-binding inhibitor and somatostatin. These results support the notion that age of onset may delineate subgroups of alcoholics with significant clinical and neurochemical differences.

Adult↗
PubMed1991

Depression among alcoholics. Relationship to clinical and cerebrospinal fluid variables.

Although depression is common among alcoholics, its determinants are poorly understood. Among 339 alcoholics, 111 (33%) had a history of major depression. Depressed, compared with never-depressed alcoholics, had a higher daily alcohol intake, more lifetime diagnoses of other anxiety and affective disorders and drug abuse, more had attempted suicide, and more reported alcoholism in both parents. Depressed alcoholics also had significantly lower cerebrospinal fluid levels of the dopamine metabolite homovanillic acid and of gamma-aminobutyric acid. Among subgroups of depressed alcoholics, secondary compared with primary depressives were more often divorced, of lower social status, with an earlier onset of alcoholism, and higher Michigan Alcohol Screening Test scores. Secondary depressives also had significantly lower cerebrospinal fluid concentrations of homovanillic acid than never depressed alcoholics. These results suggest that certain psychosocial variables, alcohol consumption, and neurochemical variables may be specifically associated with depression in alcoholics.

Adult↗
PubMed1991

Cerebrospinal fluid variables among alcoholics lack seasonal variation.

Seasonal influences on indices of serotonergic function, including cerebrospinal fluid (CSF) concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), have been reported in psychiatric patients and healthy volunteers. We examined seasonal differences in CSF concentrations of 5-HIAA among 135 alcoholics admitted to a research ward who had a lumbar puncture. No significant seasonal differences were found for either CSF concentrations of 5-HIAA or CSF concentrations of other monoamine metabolites or peptides. The possible explanations for these negative findings are discussed.

Adult↗
PubMed1990

Cerebrospinal fluid thyrotropin-releasing hormone concentrations in alcoholics and normal controls.

Alterations in hypothalamic-pituitary-thyroid axis function have been reported in alcoholism. Blunting of the thyroid-stimulating hormone (TSH) response to thyrotropin-releasing hormone (TRH) occurs in approximately 25% of alcoholic patients. Using a sensitive radioimmunoassay that allows TRH itself to be measured in cerebrospinal fluid (CSF), CSF concentrations of TRH were measured in alcoholics and normal controls. There was no significant difference in TRH concentrations between the groups. However, among the controls there was a significant correlation between CSF concentrations of the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and CSF concentrations of TRH. This correlation was lacking in the alcoholics. These findings are of interest because basic neurobiological studies have reported that TRH and serotonin are co-localized in certain neurons in the rat central nervous system.

Adult↗