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N Heintz

Publications and source records attributed to N Heintz.

At least 73 records · Page 4Linked to original sources
PubMed1991

An unusual case using DNA polymorphisms to determine parentage of human remains.

The 2-year-old daughter of two farm laborers was reported missing while the farm owner was harvesting corn. Unidentifiable tissues and body parts were subsequently found admixed with silage. Samples of blood collected from the parents of the missing child as well as portions of the tissue recovered from the silage were subjected to analysis of DNA polymorphisms with probes usually used to identify paternity. In addition, allele-specific oligonucleotides were used to detect DNA polymorphism at the DQ alpha locus following DNA amplification using the polymerase chain reaction. In this case, the DNA results established that the tissue recovered from the silage was of human origin and confirmed the probable parentage of the two farm laborers.

Accidents↗
PubMed1991

Histone gene transcription factor binding in extracts of normal human cells.

Transcriptional regulation of mammalian histone genes during S phase is achieved through activation of specific factors which interact with subtype-specific histone gene promoter sequences. It has previously been shown that in HeLa cells this induction is not mediated by obligatory changes in the DNA binding activity of histone gene transcription factors as cells progress through the cell cycle. Recently, it has been reported that the DNA binding properties of a putative histone gene transcription factor may be quite different in normal and transformed cells (J. Holthuis, T. A. Owen, A. J. van Wijnen, K. L. Wright, A. Ramsey-Ewing, M. B. Kennedy, R. Carter, S. C. Cosenza, K. J. Soprano, J. B. Lian, J. L. Stein, and G. S. Stein, Science 247:1454-1457, 1990). To determine whether the properties of well-characterized histone gene transcription factors are altered in transformed versus normal cells, we have examined the DNA binding activity of human histone transcription factors during the WI38 (a primary line of normal human fetal lung fibroblasts) cell cycle. The results demonstrate that the properties of Oct1, H4TF1, and H4TF2 are similar in WI38 and HeLa cells and that their DNA binding activities are constitutive during interphase of both normal and transformed cell lines. Although it remains possible that these factors are directly or indirectly perturbed as a result of cellular transformation, it appears unlikely that transformation results in gross changes in DNA binding activity as cells progress toward division.

Blotting, Western↗
PubMed1990

Meander tail reveals a discrete developmental unit in the mouse cerebellum.

Analyses of phenotypic mutations with altered patterns of cellular organization in invertebrate systems have lead to the identification of genes important to histogenesis. Efforts to identify genes defining segments or compartments in mammalian systems and demonstrate a role for such genes in the establishment of the cellular architectonics of the brain have been hampered by the absence of phenotypic mutations that reveal compartments. Evidence is presented that in the cerebellum of the mutant mouse, meander tail, there is an abrupt transition from the normal cytoarchitecture seen in the posterior lobes to a severely disorganized cellular pattern. Thus, in the anterior lobes the Purkinje cells are positioned randomly throughout the cortex, and other cellular layers, including the internal granule cell layer, do not form. In addition, radial Bergmann glial processes are virtually absent. One explanation for the discrete boundary of the affected area in the anterior portion of this mutant cerebellar cortex is that the meander tail gene influences compartmental cellular organization in mammalian brain.

Animals↗
PubMed1990

E1A-dependent trans-activation of the c-fos promoter requires the TATAA sequence.

Previous experiments have demonstrated that transcription of the human c-fos oncogene is activated through the action of the 289-amino acid adenovirus E1A gene product. In this study we have utilized a series of c-fos promoter deletion and substitution mutants to define regulatory sequences that allow the induction by E1A. Although the deletion of upstream promoter sequences has varying degrees of effect on overall promoter activity, these deletions retain inducibility by E1A. This includes the deletion of the serum response element and two elements that bind the ATF transcription factor. In fact, a c-fos promoter deleted to position -53, which leaves the TATA element but no other known functional element, retains inducibility, indicating a role for the TATA element in E1A control. Indeed, substitution of the c-fos TATA element (TATAA) with a TATA sequence from the simian virus 40 early promoter (TATTTAT) abolishes E1A inducibility; this promoter does retain responsiveness to cAMP induction, however, demonstrating that this TATTTAT substitution is functional. We conclude that the E1A-dependent activation of c-fos transcription is mediated through an effect on a TATA-binding protein that has specificity for the TATAA sequence.

Adenovirus Early Proteins↗
PubMed1990

Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities.

Replication of the Chinese hamster dihydrofolate reductase gene (dhfr) initiates near a fragment of stably bent DNA that binds multiple cellular factors. Investigation of protein interactions with the dhfr bent DNA sequences revealed a novel nuclear protein that also binds to domain B of the yeast origin of replication, the autonomously replicating sequence ARS1. The origin-specific DNA-binding activity was purified 9,000-fold from HeLa cell nuclear extract in five chromatographic steps. Protein-DNA cross-linking experiments showed that a 60-kDa polypeptide, which we call RIP60, contained the origin-specific DNA-binding activity. Oligonucleotide displacement assays showed that highly purified fractions of RIP60 also contained an ATP-dependent DNA helicase activity. Covalent radiolabeling with ATP indicated that the DNA helicase activity resided in a 100-kDa polypeptide, RIP100. The cofractionation of an ATP-dependent DNA helicase with an origin-specific DNA-binding activity suggests that RIP60 and RIP100 may be involved in initiation of chromosomal DNA synthesis in mammalian cells.

Adenosine Triphosphatases↗
PubMed1989

Histone H1 subtype-specific consensus elements mediate cell cycle-regulated transcription in vitro.

In this study we used nuclear extracts from centrifugally elutriated cell populations to study histone H1 transcriptional regulation during the cell cycle. Analysis of mutations within the H1 promoter establish that both of the H1 subtype-specific consensus elements participate in induction of transcription upon entry into S phase. The DNA binding activity of H1TF2, which specifically interacts with the H1 proximal subtype-specific element, is increased in S-phase nuclear extracts, whereas no increase in DNA binding is observed for the H1 distal subtype-specific DNA transcription factor H1TF1 or the H2b subtype-specific factor OTF1. These data strongly support the idea that histone gene subtype-specific transcription factors are important for S-phase-dependent expression of histone genes. Further studies of these factors will be important for increased understanding of the transition from G1 to S phase of the mammalian cell cycle.

Animals↗
PubMed1989

Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element.

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.

Binding Sites↗
PubMed1988

Coxsackievirus B-3-induced myocarditis. Virus and actinomycin D treatment of myocytes induces novel antigens recognized by cytolytic T lymphocytes.

BALB/c mice inoculated with 6 x 10(4) plaque-forming units of a myocarditic variant of Coxsackievirus, group B, type 3 (CVB3M) developed three distinct CTL populations recognizing putative virion (virus specific CTL, VCTL), normally expressed heart (autoreactive CTL, ACTL), and aberrant metabolic (MCTL) Ag. Induction of the MCTL-specific Ag on cardiocytes correlated with the ability of the myocarditic viruses and actinomycin D to interfere with cellular metabolism as measured by 3H-uridine and 3H-leucine incorporation. ACTL specifically lysed uninfected cardiocyte targets, but ACTL Ag expression was lost soon after infection of monocytes only to reappear 6 h later. MCTL and VCTL could be separated by adsorption to myocytes treated with either actinomycin D- or UV-inactivated CVB3M. MCTL cross-reactively lysed myocytes infected with another virus suppressing 3H-uridine incorporation (encephalomyocarditis virus) but failed to react to targets infected with viruses not inhibiting cell metabolism. VCTL specifically lysed only cells infected with the homologous virus. ACTL belonged to the CD8 (Lyt-2+) T cell subset, whereas both VCTL and MCTL were CD4 (L3T4+) T cells.

Animals↗
PubMed1988

Transcription factor OTF-1 is functionally identical to the DNA replication factor NF-III.

Octamer transcription factor-1 (OTF-1) and nuclear factor III (NF-III) are sequence-specific DNA binding proteins that activate transcription and DNA replication, respectively. It is shown here that OTF-1 is physically and biologically indistinguishable from NF-III. This conclusion is based on the following observations. First, the two proteins have identical mobilities by SDS-polyacrylamide gel electrophoresis. Second, OTF-1 binds to the adenovirus origin of DNA replication at the same site and with the same affinity as NF-III. Third, OTF-1 can substitute for NF-III in activating the initiation of adenovirus DNA replication in vitro. Fourth, the ability of OTF-1 to stimulate viral DNA replication is dependent on the presence of an intact NF-III binding site within the origin of replication. Fifth, NF-III can substitute for OTF-1 in activating in vitro transcription from the human histone H2b promoter. These data suggest the possibility that NF-III/OTF-1 is a protein that functions in both cellular DNA replication and transcription.

Adenoviridae↗
PubMed1988

Definition of multiple, functionally distinct TATA elements, one of which is a target in the hsp70 promoter for E1A regulation.

We have dissected the human hsp70 promoter to define sequence elements allowing response to E1A. Alterations of sequence upstream of the TATA element, either with Bal 31 nuclease or by site-directed mutagenesis, had little or no effect on the response of the promoter to E1A. In general, the basal level was reduced, indicating that these sites interact with factors important for transcription, but regulation persisted. Although a CAT gene driven by just the hsp70 TATA (void of upstream sequences) could be stimulated by E1A, a similar construct containing the early SV40 TATA element was not. Analysis of several additional such constructions indicated that the specific sequence TATAA was crucial. Substitution of the TATAA sequence with the SV40 TATTTAT element in the context of the wild-type hsp70 promoter resulted in loss of E1A inducibility, but maintenance of heat inducibility. Replacement of this element with sequences not related to any TATA element resulted in loss of activity and inducibility. Thus, the SV40 TATA equivalent is functional in the context of the hsp70 promoter but cannot be induced by E1A. We conclude that the target for E1A induction of the hsp70 promoter is TATAA, and that multiple functionally distinct TATA elements, and presumably cognate transcription factors, can be distinguished in eukaryotic cells.

Bacteriophage lambda↗
PubMed1988

Cell type-specific expression of a human histone H1 gene.

The expression of a human histone H1 gene (Hh8c H1) was studied in several different human cell lines. Nuclease S1 analysis revealed that this gene is expressed at high levels in KB cells and that it is regulated during the cell cycle, but that it is not expressed in several other human cell lines (HeLa, HEK, 293, and Hep G2). The differential expression of the Hh8c H1 gene in vivo was not due to gross chromosomal rearrangements or to activation of the entire histone gene cluster at this genomic locus. Thus, the cell type-specific expression of this human H1 gene must result from either critical differences in the promoter sequences of this gene in KB and HeLa cells, or from cell-specific utilization of trans-acting transcription factors.

Cells, Cultured↗
PubMed1988

Cell-cycle regulation of a human histone H2b gene is mediated by the H2b subtype-specific consensus element.

Mammalian histone gene transcription is increased approximately fivefold during the transition from the G1 phase to the S phase of the cell cycle. In this study, we present a detailed in vivo analysis of the human histone H2b promoter, which establishes that transcriptional regulation of this gene is mediated by a subtype-specific consensus element containing the core octanucleotide ATTTGCAT. Our results demonstrate that the activity of this sequence is specific for S phase. Comparative analysis of different replication variant mammalian histone gene promoters and our knowledge of the transcription factors interacting with the human histone H2b and H4 promoters allow us to conclude that coordinate regulation of histone gene transcription in higher eukaryotes is mediated by distinct factors. We propose a simple model for transcriptional regulation of mammalian histone gene expression, which incorporates both the distinct features of the individual histone gene promoters and the apparent functional equivalence of the specific sequence elements regulating transcription of each histone gene subtype.

Aphidicolin↗
PubMed1988

Purification of the human histone H4 gene-specific transcription factors H4TF-1 and H4TF-2.

Transcription of the cell-cycle-regulated human histone genes increases approximately fivefold during S phase. One step toward the elucidation of the biochemical mechanisms that govern cell-cycle-regulated expression of these genes is to purify and characterize the transcription factors that regulate these promoters. Here, we describe the purification of two previously identified factors, H4TF-1 and H4TF-2, which bind the human histone H4 promoter. Purification was achieved through a combination of ion-exchange and oligonucleotide affinity chromatography. On the basis of analysis of purified fractions by SDS-polyacrylamide gels and UV cross-linking, we believe that H4TF-1 is two polypeptides of 105 and 110 kD. This factor binds to a GC-rich DNA sequence required for maximal expression of the H4 gene but does not bind to any Sp1 consensus elements tested. H4TF-2 is a 65-kD protein that binds specifically to sequences within that highly conserved H4 subtype-specific consensus promoter element. Both highly purified factors activated transcription in vitro only from H4 promoters that contained their binding sequences, demonstrating that H4TF-1 and H4TF-2 are H4-specific transcription factors that potentiate expression of this gene.

Base Sequence↗
PubMed1987

Purification and characterization of OTF-1, a transcription factor regulating cell cycle expression of a human histone H2b gene.

An octamer-binding transcription factor, OTF-1, which stimulates transcription of a human histone H2b gene, has been purified from HeLa nuclear extracts. This purification was achieved through the use of DNA affinity chromatography, and the factor was unambiguously identified by renaturation of activity following SDS-polyacrylamide gel electrophoresis. The purified factor retained the ability to efficiently stimulate H2b transcription in a reconstituted in vitro system. This effect was dependent upon an intact octamer element and was observed in the absence of the other H2b promoter elements (except the TATA motif). Furthermore, this activity was not detected in nuclear extracts prepared from cells synchronized in G2, in agreement with the in vivo data showing S-phase-specific utilization of the octamer element. From these data, we conclude that we have purified the bona fide H2b transcriptional regulatory factor.

Cell Cycle↗
PubMed1987

Sequences controlling histone H4 mRNA abundance.

The post-transcriptional regulation of histone mRNA abundance is manifest both by accumulation of histone mRNA during the S phase, and by the rapid degradation of mature histone mRNA following the inhibition of DNA synthesis. We have constructed a comprehensive series of substitution mutants within a human H4 histone gene, introduced them into the mouse L cell genome, and analyzed their effects on the post-transcriptional control of the H4 mRNA. Our results demonstrate that most of the H4 mRNA is dispensable for proper regulation of histone mRNA abundance. However, recognition of the 3' terminus of the mature H4 mRNA is critically important for regulating its cytoplasmic half-life. Thus, this region of the mRNA functions both in the nucleus as a signal for proper processing of the mRNA terminus, and in the cytoplasm as an essential element in the control of mRNA stability.

Animals↗
PubMed1987

Control of hsp70 RNA levels in human lymphocytes.

The expression of a hsp70 gene in human cells has previously been shown to be related to the growth state of the cells. As an alternative to in vitro synchronization procedures, we have measured steady-state levels of the RNA for a heat-shock protein 70 (hsp70) in human peripheral blood mononuclear cells (PBMC) that are naturally quiescent in a G0 state. The probe used recognized, on RNA blots, one single band. The levels of this hsp70 RNA are elevated in circulating PBMC and decrease when the cells are incubated with serum, or phytohemagglutinin, or simply when they are incubated in culture medium. The levels of hsp70 RNA decrease within 30 min after in vitro culture, and are accompanied by an increase in the levels of c-fos RNA. These findings, together with other recent reports in the literature, suggest a possible role of the hsp70 proteins in the regulation of cell growth.

Cell Line↗