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PubMed1992

[Chiasma distribution in the lampbrush chromosomes of the chicken Gallus gallus domesticus: hot spots of recombination and their possible role in proper dysjunction of homologous chromosomes at the first meiotic division].

Chiasma distribution in the lambrush chromosomes of the chicken Gallus gallus domesticus was studied. The data of the authors show that the general pattern of chiasmata in the interstitional region of chromosomes corresponds to the Poisson distribution. However, in the telomeric and subtelomeric regions of all chicken macrochromosomes one can see chiasma as a rule. In the half of 140 microchromosomes from 24 different oocytes, there are also the telomeric chiasmata. On the basis of this observation, it may be predicted that there are hot spots of recombination near or into the telomeric GC-rich heterochromatic bands of chicken chromosomes. We suggest that these hot spots of recombination near the telomeres are a necessary facility for not only macrochromosomes but all microchromosomes as well to have at least one chiasma. The constant presence of at least one chiasma in a bivalent in needed for correct disjunction of homologous chromosomes at the first meiotic division.

Animals↗
PubMed1991

Genomic structure of the human prion protein gene.

Creutzfeld-Jacob disease and Gerstmann-Sträussler syndrome are rare degenerative disorders of the nervous system which have been genetically linked to the prion protein (PrP) gene. The PrP gene encodes a host glycoprotein of unknown function and is located on the short arm of chromosome 20, a region with few known genes or anonymous markers. The complete structure of the PrP gene in man has not been determined despite considerable interest in its relationship to these unusual disorders. We have determined that the human PrP gene has the same simple genomic structure seen in the hamster gene and consists of two exons and a single intron. In contrast to the hamster PrP gene the human gene appears to have a single major transcriptional start site. The region immediately 5' of the transcriptional start site of the human PrP gene demonstrates the GC-rich features commonly seen in housekeeping genes. Curiously, the genomic clone we have isolated contains a 24-bp deletion that removes one of five octameric peptide repeats predicted to form a B-pleated sheet in this region of the PrP. We have also identified 5' of the PrP gene an RFLP which has a high degree of heterozygosity and which should serve as a useful marker for the pter-12 region of human chromosome 20.

Amino Acid Sequence↗
PubMed1992

The parathyroid hormone-related protein (PTHrP) gene preferentially utilizes a GC-rich promoter and the PTHrP 1-139 coding pathway in normal human amnion.

Parathyroid hormone-related protein (PTHrP) is an oncofetal protein that is widely expressed in mammalian tissues. The PTHrP gene is a complex with three transcriptional start-sites, two TATA boxes and a GC-rich region, and three predicted polypeptide products, PTHrP 1-141, PTHrP 1-139, and PTHrP 1-173. The originally discovered form of PTHrP, PTHrP 1-141, and the classical TATA box promoters are generally assumed to be the major pathways of PTHrP gene expression. We have used the polymerase chain reaction (PCR) to study PTHrP gene expression in the human amnion. Our studies demonstrate that the GC-rich promoter is preferentially used and that PTHrP 1-139 is the major PTHrP mRNA expressed in human amnion. PTHrP 1-139 lacks the carboxy-terminal arginine and histidine residues of PTHrP 1-141; these two basic amino acids could have significant effects on the biological activity of PTHrP. These preferential pathways for PTHrP gene expression are shared by malignant and normal human tissues.

Amnion↗
PubMed1992

Cloning and characterization of the 5'-flanking region of the human topoisomerase II alpha gene.

Topoisomerases are essential enzymes for DNA metabolism in prokaryotes and eukaryotes. In human cells, DNA topoisomerase II enzyme activity can be modulated by both viral transformation and changes in proliferation status. To identify elements important for regulation of topoisomerase II alpha gene expression, genomic DNA clones covering the 5'-end of the gene were isolated. The intron/exon structure of a 2.5-kilobase region encompassing the translation start site was determined. Transcription was found to initiate at multiple sites clustered around 90 base pairs 5' to the ATG initiation codon. Transient expression of chimeric topoisomerase II-reporter gene constructs in HeLa cells revealed that the 5'-flanking region exhibited promoter activity. The region -90 to -1 upstream of the major transcription start site was shown by deletion analysis to include a promoter. This minimal promoter lacks a TATA box, is moderately GC-rich, and contains a high frequency of CpG dinucleotides; characteristic of a "housekeeping" gene promoter. Maximal promoter activity was observed using a fragment extending to position -562. Putative regulatory elements are contained within and immediately upstream of the minimal promoter region. The regulatory region of the topoisomerase II alpha gene identified here is similar in basic structure to those of the human thymidine kinase and DNA polymerase alpha genes, which are also controlled by proliferation-specific factors.

Base Sequence↗
PubMed1992

Triplet repeat mutations in human disease.

Triplet repeats are the sites of mutation in three human heritable disorders, spinal and bulbar muscular atrophy (SBMA), fragile X syndrome, and myotonic dystrophy (DM). These repeats are GC-rich and highly polymorphic in the normal population. Fragile X syndrome and DM are examples of diseases in which premutation alleles cause little or no disease in the individual, but give rise to significantly amplified repeats in affected progeny. This newly identified mechanism of mutation has, so far, been identified in two of the most common heritable disorders, fragile X syndrome and DM, and one rare disease, SBMA.

Female↗
PubMedDate not supplied

Polymerase chain reaction analysis of fragile X mutations.

The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals.

Base Sequence↗
PubMed1992

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

Animals↗
PubMed1992

A novel myoblast enhancer element mediates MyoD transcription.

The MyoD gene can orchestrate the expression of the skeletal muscle differentiation program. We have identified the regions of the gene necessary to reproduce transcription specific to skeletal myoblasts and myotubes. A proximal regulatory region (PRR) contains a conserved TATA box, a CCAAT box, and a GC-rich region that includes a consensus SP1 binding site. The PRR is sufficient for high levels of skeletal muscle-specific activity in avian muscle cells. In murine cells the PRR alone has only low levels of activity and requires an additional distal regulatory region to achieve high levels of muscle-specific activity. The distal regulatory region differs from a conventional enhancer in that chromosomal integration appears necessary for productive interactions with the PRR. While the Moloney leukemia virus long terminal repeat can enhance transcription from the MyoD PRR in both transient and stable assays, the simian virus 40 enhancer cannot, suggesting that specific enhancer-promoter interactions are necessary for PRR function.

Animals↗
PubMed1991

The expression of cathepsin B and other lysosomal proteinases in normal tissues and in tumors.

The mRNA for the lysosomal proteinases cathepsins B, D, H, L, and S are broadly distributed in normal rodent tissues. Although total cathepsin mRNA levels generally parallel the protein catabolic activity of the tissues, the expressions of the individual enzymes do not appear to be linked. Thus, the relative proportions of the individual messages are found to vary from tissue to tissue. Further evidence for the independent regulation of lysosomal proteinase expression is derived from observations of selective increases in mRNA levels for individual proteinases in rodent tumors. Only cathepsin B mRNA is elevated in a highly metastatic murine B16a melanoma and in a Walker-256 rat carcinosarcoma, while Moloney murine sarcoma virus-transformed fibroblasts express increased mRNA for cathepsins B, D, and L and normal levels for H and S. To address the regulation of cathepsin B expression, the mouse cathepsin B gene and its 5'-upstream region were cloned. The gene has 10 exons and 9 introns spanning about 20 kilobases. The 5'-upstream region and exon 1 are GC-rich with several potential Sp1 binding sites. TATA and CAAT motifs adjacent to the transcription start site are not evident. These properties are characteristic of mammalian "housekeeping" genes. B16 melanoma cells contain three cathepsin B transcripts of 2.2, 4.0 and 5.0 kilobases. The two larger messages, which were not found in normal tissues, contain unusually long 3'-untranslated regions resulting from the alternative cleavage and polyadenylation of the 3' end of the cathepsin B pre-mRNA in B16 melanomas. As all three messages encoded normal preprocathepsin B, cathepsin B secretion by melanoma cells is probably due to posttranslational mechanisms and not to alternative splicing or gene mutation.

Animals↗
PubMed1992

Molecular and functional analysis of the XPBC/ERCC-3 promoter: transcription activity is dependent on the integrity of an Sp1-binding site.

The human XPBC/ERCC-3 gene, which corrects the excision-repair defect in xeroderma pigmentosum group B cells and the UV-sensitive CHO mutant 27-1 cells, appears to be expressed constitutively in various cell types and tissues. We have analysed the structure and functionality of the XPBC/ERCC-3 promoter. Transcription of the XPBC/ERCC-3 gene is initiated from heterogeneous sites, with a major startpoint mapped at position -54 (relative to the translation start codon ATG). The promoter region does not possess classical TATA and CAAT elements, but it is GC-rich and contains three putative Sp1-binding sites. In addition, there are two elements related to the cyclic AMP (cAMP)-response element (CRE) and the 12-O-tetradecanoyl phorbol-13-acetate-response element (TRE) in the 5'-flanking region. Transient expression analysis of XPBC/ERCC-3 promoter-CAT chimeric plasmids revealed that a 127-bp fragment, spanning position -129 to -3, is minimally required for the promoter activity. Transcription of the XPBC/ERCC-3 promoter depends on the integrity of a putative Sp1-binding site in close proximity to the major cap site. Band shift assays showed that this putative Sp1-binding site can interact specifically with a nuclear factor, most likely transcription factor Sp1 (or an Sp1-like factor) in vitro.

Animals↗
PubMed2008

TAp73beta and DNp73beta activate the expression of the pro-survival caspase-2S.

p73, the p53 homologue, exists as a transactivation-domain-proficient TAp73 or deficient deltaN(DN)p73 form. Expectedly, the oncogenic DNp73 that is capable of inactivating both TAp73 and p53 function, is over-expressed in cancers. However, the role of TAp73, which exhibits tumour-suppressive properties in gain or loss of function models, in human cancers where it is hyper-expressed is unclear. We demonstrate here that both TAp73 and DNp73 are able to specifically transactivate the expression of the anti-apoptotic member of the caspase family, caspase-2(S). Neither p53 nor TAp63 has this property, and only the p73beta form, but not the p73alpha form, has this competency. Caspase-2 promoter analysis revealed that a non-canonical, 18 bp GC-rich Sp-1-binding site-containing region is essential for p73beta-mediated activation. However, mutating the Sp-1-binding site or silencing Sp-1 expression did not affect p73beta's transactivation ability. In vitro DNA binding and in vivo chromatin immunoprecipitation assays indicated that p73beta is capable of directly binding to this region, and consistently, DNA binding p73 mutant was unable to transactivate caspase-2(S). Finally, DNp73beta over-expression in neuroblastoma cells led to resistance to cell death, and concomitantly to elevated levels of caspase-2(S.) Silencing p73 expression in these cells led to reduction of caspase-2(S) expression and increased cell death. Together, the data identifies caspase-2(S) as a novel transcriptional target common to both TAp73 and DNp73, and raises the possibility that TAp73 may be over-expressed in cancers to promote survival.

Binding Sites↗
PubMed1992

A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins.

The KAR2 gene of Saccharomyces cerevisiae codes for an essential chaperone protein (BiP) that is localized in the lumen of the endoplasmic reticulum (ER). The high basal rate of transcription of KAR2 is increased transiently by heat shock: prolonged induction occurs when unfolded proteins accumulate in the ER. Three cis-acting elements in the KAR2 promoter control expression of KAR2: (i) a GC-rich region that contributes to the high level of constitutive expression, (ii) a functional heat shock element (HSE) and (iii) an element (UPR) that is involved in the induction of BiP mRNA by unfolded proteins. By analyzing internal deletion mutants of the KAR2 promoter, we demonstrate here that these three elements regulate transcription of KAR2 independently. Furthermore, the 22 bp UPR element causes a heterologous (CYC1) promoter to respond to the presence of unfolded proteins in the ER. Extracts of both stressed and unstressed yeast cells contain proteins that bind specifically to synthetic HSE and UPR elements and retard their migration through gels. Binding proteins specific for the UPR element can be fractionated by ammonium sulfate precipitation. Two of the proteins UPRF-1 and UPRF-2 (which is apparently a proteolytic degradation product of UPRF-1) bind inefficiently to mutant versions of the UPR that are unable to confer responsiveness to unfolded proteins to the (CYC1) promoter. UPRF-1 therefore displays the properties expected of a transcription factor that is involved in the sustained response of the KAR2 promoter to unfolded proteins in the ER. These experiments show that yeast cells can activate a transcription factor that stimulates expression of a nuclear gene in response to the accumulation of unfolded proteins in another cellular compartment.

Base Sequence↗
PubMed1991

Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.

We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.

Base Sequence↗
PubMed1979

Variable substructure in the secondary constriction of the human chromosome 1.

The secondary constriction in human chromosome 1 consists of a proximal segment stained by the GC-specific fluorochrome mithramycin and a distal segment stained by such fluorochromes as DAPI or DIPI, which show enhanced fluorescence intensities in AT-rich regions of the chromosomes. A study involving 21 individuals revealed that both parts are independently involved in length variability. In two cases, two GC-rich regions separated by an AT-rich segment and an additional distal AT-rich part were found.

Base Sequence↗
PubMed1992

Compositional bimodality and evolution of retroviral genomes.

The compositional distributions of genomes, genes (and their third codon positions) and long terminal repeats from retroviruses of warm-blooded vertebrates are characterized by a striking bimodality which is accompanied by a remarkable compositional homogeneity within each retroviral genome. A first, major class of retroviral genomes is GC-rich, whereas a second, minor class is GC-poor. Representative expressed viral genomes from the two classes integrate in GC-rich and GC-poor isochores, respectively, of host genomes. The first class comprises all oncoviruses (except B-types and some D-types), the second, lentiviruses, spumaviruses, as well as B-type and some D-type oncoviruses (e.g., mouse mammary tumor virus and simian retroviruses type D, respectively). The compositional bimodal distribution of retroviral genomes and the accompanying compositional homogeneity within each retroviral genome appear to be the result of the compositional evolution of retroviral genomes in their integrated form.

Animals↗
PubMed1991

The structure of the gene coding for the mouse cell adhesion molecule uvomorulin.

We have recently shown that the Ca2+ dependent cell adhesion molecule uvomorulin is encoded by a single gene, localized on mouse chromosome 8. Here we describe the organization of the uvomorulin gene and give an initial characterization of the uvomorulin promoter. Uvomorulin is encoded by 16 exons, which are distributed over a region of more than 40 kb genomic DNA. The exon structure of the genes for uvomorulin and its chicken homologue L-CAM are nearly identical and thus highly conserved. The relationship between the exon structure and the structure of the uvomorulin protein is analysed. The initiation site of transcription of the uvomorulin gene is located 127 bp upstream of the translation start site in a GC-rich region with no TATA-box, but with a GC-box in position -48 and a CCAAT-box starting at position -65 with respect to the transcription start site. 1.6 kb of the uvomorulin promoter (-1492 to + 92) confer cell type specific promoter activity to the CAT reporter gene. Homologies to known cis acting elements of other promoters are discussed.

3T3 Cells↗
PubMed1975

Denaturation mapping studies on the circular chloroplast deoxyribonucleic acid from pea leaves.

The structure of circular pea chloroplast DNA (ctDNA) has been analyzed by denaturation mapping. All of the pea ctDNA molecules that were examined had identical gross base sequences. Denaturation maps were constructed at denaturation levels of 2.5%, 22%, and 44%. These denaturation maps showed that the circular pea ctDNA contained six small AT-rich regions on one-half of the DNA molecule, and two small GC-rich regions on the other half of the DNA molecule. The structure of pea ctDNA circular dimers was also examined. The results showed that the pea ctDNA circular dimers consisted of two monomer length units integrated in tandem repeat.

Chloroplasts↗
PubMed1992

Structure and developmental regulation of the B-lymphoid tyrosine kinase gene blk.

The murine blk gene, which encodes a B-lymphoid-specific tyrosine kinase of the Src family (p55blk), contains 13 exons that span more than 30 kilobases of DNA on chromosome 14. In the first three exons, which encode the 5'-untranslated region and N-terminal amino acid sequence unique to p55blk, the blk gene differs from other members of the src family; in the last 10 exons, the organization of the blk gene is similar to that of other src genes. By primer extension and S1 nuclease protection analyses, we show that blk transcripts initiate from four major sites at the 5'-flank of blk; two sites predominate. The resulting transcripts differ only in the lengths of their 5'-untranslated sequences and encode identical proteins. None of the transcriptional start sites are preceded by consensus TATA elements, AT-rich elements, or extensive GC-rich regions. Expression of blk is regulated during B-cell development: blk RNA is expressed in all pro-B-, pre-B-, and mature B-cell lines examined, but is absent from plasma cell lines. Immunolocalization of p55blk in normal mouse spleen supports these observations: staining is restricted to lymphocytes and is concentrated in regions rich in B-cells; plasma cells and stromal cells are not stained with anti-Blk antibodies. Assays for RNA synthesis in isolated nuclei indicate that the lineage and developmental stage specificities of blk expression are regulated at least in part by changes in its rate of transcription.

Amino Acid Sequence↗