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At least 919 records · Page 51Linked to original sources
PubMed1988

Extinction of growth hormone expression in somatic cell hybrids involves repression of the specific trans-activator GHF-1.

Growth hormone (GH) expression in pituitary-derived cells has been attributed to the presence of a positive trans-activator, GHF-1, which binds to two sites on the GH promoter. Somatic cell hybridization of non-GH-expressing L cells with pituitary-derived GH3 cells usually results in extinction of GH production. While previous studies showed that extinction occurs at the level of GH transcription, the exact mechanism remained elusive. We therefore characterized two parental cell lines and three hybrids, two of which extinguish GH expression and one in which GH is reexpressed after loss of mouse chromosomal material. Using in vivo transfections, in vitro transcription, DNAase I footprints, and immunoblotting experiments, no evidence for a direct repressor of GH transcription was found. Rather, extinction of GH expression in fibroblast x pituitary hybrids was accompanied by loss of GHF-1 protein and mRNA expression, suggesting that extinction occurs by repression of this trans-activator.

Animals↗
PubMed1989

A single amino acid can determine the DNA binding specificity of homeodomain proteins.

Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox. This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins. We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd). This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd). We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd. This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins. We further show that Prd contains two DNA binding activities. The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.

Amino Acid Sequence↗
PubMed1989

Intramolecular transposition by Tn10.

Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.

Base Sequence↗
PubMed1989

Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit.

The minimal DNA sequence required for the formation of a stable complex with Drosophila heat shock factor (HSF) in vitro is an inverted repeat of a 5 bp recognition unit, -GAA-. Surprisingly, both permutations of this 5 bp unit, head-to-head and tail-to-tail, bind to HSF with similar affinity and with striking 2-fold symmetry. HSF also binds to longer arrays of inverted 5 bp units, and the size of the HSF footprint increases with the addition of each 5 bp unit to these arrays. However, the electrophoretic mobility of the HSF-DNA complexes decreases most distinctly with the addition of every three 5 bp units. Cross-linking of purified HSF in the absence of DNA generates complexes with the sizes expected of HSF trimers. We propose that trimers of HSF bind to DNA and that the number of HSF subunits in direct contact with DNA is determined by the number of correctly positioned 5 bp recognition units.

Animals↗
PubMed1990

Nucleosome positioning modulates accessibility of regulatory proteins to the mouse mammary tumor virus promoter.

Minichromosomes containing the MMTV hormone responsive element (HRE) exhibit precisely positioned nucleosomes. Chromatin reconstitution of short HRE DNA fragments also results in precise positioning of nucleosomes as revealed by footprinting, which suggests that information for nucleosome phasing is contained within this short sequence. While hormone receptors bind naked DNA and reconstituted nucleosomes with similar affinities (3- to 5-fold difference), NFI, a transcription factor essential for efficient utilization of the MMTV promoter, binds naked DNA very tightly but does not bind the nucleosomally organized promoter. Hormone receptor binding to the MMTV nucleosome does not dissociate the nucleosome but leads to greater accessibility of the promoter-proximal end to exonuclease III. Precise positioning of one nucleosome over the MMTV promoter could repress transcription by preventing NFI binding in the absence of hormone, while still allowing interaction of activated hormone receptor with HRE.

Animals↗
PubMed1990

Destruction of U2, U4, or U6 small nuclear RNA blocks trans splicing in trypanosome cells.

We have used permeable cells of Trypanosoma brucei to analyze the role of snRNAs in the trans splicing process. Degradation of U2, U4, or U6 snRNA by site-directed cleavage with complementary deoxyoligonucleotides and RNAase H inhibits trans splicing of the spliced leader (SL) RNA and newly synthesized alpha-tubulin pre-mRNAs. Cleavage of U snRNAs abolishes the appearance of putative trans splicing reaction intermediates and products, namely, linear branched molecules consisting of the SL intron joined to high molecular weight RNA (Y structures) and free SL intron. This indicates that U snRNAs are required for an early step in trans splicing. alpha-tubulin transcripts synthesized in the absence of trans splicing are unstable, suggesting that the addition of the SL sequence stabilizes pre-mRNAs against degradation. Our results provide direct evidence for the participation of U2 and U4/U6 snRNPs in trans splicing.

Animals↗
PubMed1990

Demonstration in living cells of an intragenic negative regulatory element within the rodent c-fos gene.

We studied c-fos gene expression in rat fibroblasts by microinjection of regulatory DNA sequences, such as the serum response element (SRE) present in c-fos promotor, in order to compete directly with such sequences for binding of putative regulatory factors. We show that an additional fos intragenic regulatory element (FIRE) is located at the end of exon 1. When coinjected with an SRE oligonucleotide, it induced c-fos expression in quiescent cells, whereas injection of SRE sequence alone failed to do so. Moreover, injection in quiescent cells of an SRE oligonucleotide together with a p-fos-lacZ construct containing the c-fos SRE as well as an in-frame insertion of FIRE resulted in a block to beta-galactosidase expression that can be relieved by coinjection of the FIRE sequence.

Animals↗
PubMed1990

CNS stem cells express a new class of intermediate filament protein.

Multipotential CNS stem cells receive and implement instructions governing differentiation to diverse neuronal and glial fates. Exploration of the mechanisms generating the many cell types of the brain depends crucially on markers identifying the stem cell state. We describe a gene whose expression distinguishes the stem cells from the more differentiated cells in the neural tube. This gene was named nestin because it is specifically expressed in neuroepithelial stem cells. The predicted amino acid sequence of the nestin gene product shows that nestin defines a distinct sixth class of intermediate filament protein. These observations extend a model in which transitions in intermediate filament gene expression reflect major steps in the pathway of neural differentiation.

Amino Acid Sequence↗
PubMed1990

Trans splicing of nematode pre-messenger RNA in vitro.

In nematodes, a fraction of mRNAs contains a common 22 nucleotide 5' terminal spliced leader (SL) sequence derived by trans splicing. Here, we show that a cell-free extract prepared from developing embryos of the parasitic nematode Ascaris lumbricoides catalyzes accurate and efficient SL addition to a synthetic pre-mRNA at an authentic trans splice acceptor site. SL addition occurs via a trans splicing reaction that proceeds through Y-branched intermediates. The branchpoint is located at either of two adenosine residues located 18 and 19 nucleotides upstream of the splice acceptor site.

Animals↗
PubMed1991

Translational stimulation: RNA sequence and structure requirements for binding of Com protein.

Translation of the bacteriophage Mu mom gene is positively regulated by the phage Com protein. We report here that purified Com protein specifically stimulates mom gene expression in vitro. Furthermore, Com is shown to bind a site in the mom translational initiation region (TIR) in a sequence-specific manner. In vitro RNA footprint experiments have been used to define the Com-binding site and to study mRNA secondary structure in the mom TIR. Com binding is shown to correlate with a conformational change in the mom TIR both in vivo and in vitro. The role of secondary structure was further examined by testing the effects of mutations in the TIR on translation and stimulation. The results support a model for translational stimulation in which Com binding induces a conformational change in the mom mRNA, thereby enhancing ribosome binding.

Bacteriophage mu↗
PubMed1991

The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules.

Kallmann syndrome associates hypogonadotropic hypogonadism and anosmia and is probably due to a defect in the embryonic migration of olfactory and GnRH-synthesizing neurons. The Kallmann gene had been localized to Xp22.3. In this study 67 kb of genomic DNA, corresponding to a deletion interval containing at least part of the Kallmann gene, were sequenced. Two candidate exons, identified by multiparameter computer programs, were found in a cDNA encoding a protein of 679 amino acids. This candidate gene (ADMLX) is interrupted in its 3' coding region in the Kallmann patient, in which the proximal end of the KAL deletion interval was previously defined. A 5' end deletion was detected in another Kallmann patient. The predicted protein sequence shows homologies with the fibronectin type III repeat. ADMLX thus encodes a putative adhesion molecule, consistent with the defect of embryonic neuronal migration.

Amino Acid Sequence↗
PubMed1992

Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system.

We have generated mice carrying a mutation of the gene encoding the low affinity NGF receptor p75NGFR by targeted mutation in embryonic stem cells. Mice homozygous for the mutation were viable and fertile. Immunohistochemical analyses of the footpad skin of mutant mice revealed markedly decreased sensory innervation by calcitonin gene-related peptide- and substance P-immunoreactive fibers. The defective innervation was correlated with loss of heat sensitivity and associated with the development of ulcers in the distal extremities. Complicated by secondary bacterial infection, the ulcers progressed to toenail and hair loss. Crossing a human transgene encoding p75NGFR into the mutant animals rescued the absent heat sensitivity and the occurrence of skin ulcers and increased the density of neuropeptide-immunoreactive sensory innervation of footpad skin. The mutation in the gene encoding p75NGFR did not decrease the size of sympathetic ganglia or the density of sympathetic innervation of the iris or salivary gland. Our results suggest that p75NGFR has an important role in the development and function of sensory neurons.

Animals↗
PubMed1991

Rapid genetic analysis of families with polycystic kidney disease 1 by means of a microsatellite marker.

Presymptomatic diagnosis of polycystic kidney disease 1 (PKD1) is possible by genetic linkage analysis with markers from both sides of the disease locus. The existing proximal markers are not informative in many families, so such analysis is difficult and time-consuming. We sought more useful length polymorphisms on the proximal side of the locus among simple sequence repeats (microsatellites). We identified two microsatellite polymorphisms that lie closer to the PKD1 locus than any previously described highly variable marker. One, SM7, is especially informative; we have found fourteen alleles and the observed heterozygosity in caucasians is 62.7%. Genetic linkage analysis in PKD1 families suggests that both of the markers lie proximal to the disease gene, closer than existing flanking markers. These polymorphisms can be simply assayed by polymerase chain reaction amplification of the variable regions, which generates DNA fragments that can be separated on non-denaturing acrylamide gels and directly examined after gel staining. This rapid, inexpensive, and non-radioactive method of linkage analysis allows the complete study of DNA samples within 8 h.

Alleles↗
PubMed1988

Differences in DNA-fingerprints between remission and relapse in childhood acute lymphoblastic leukemia.

DNA "fingerprint" (DNA-F) analysis, based on the polymorphism caused by numeric variations in the tandem repeats of minisatellite areas of the human genome, has a potential capacity to reveal even minor genomic changes. In this study we have applied DNA-F to the detection of residual disease in leukemia. In order to identify normal and leukemic cell populations, we used two molecular probes: Jeffrey's minisatellite probes and M13 wild type phage probe, which detect different sets of polymorphic fragments in the human genome. Comparison of varying minisatellite fragments between remission and relapse was performed by Southern blot hybridization in seven patients with acute lymphoblastic leukemia (ALL). The results suggest that Southern hybridization with DNA "fingerprint" probes can prove to be a sensitive method in the detection of minimal residual disease in ALL.

Adolescent↗
PubMed1980

Persistence and spread of a chloramphenicol resistance-mediating plasmid in antigenic types of Escherichia coli, pathogenic for piglets.

All chloramphenicol-resistant Escherichia coli strains isolated from piglets in the State veterinary Serum Laboratory, Copenhagen, in 1974-1975 harbored plasmids of IncFII group with largely the same resistance markers. Two strains from 1978 carried plasmids with similar characters. Restriction enzyme analysis of DNA from these plasmids with restriction endonucleases EcoRI, BglII, and PstI shows that the Cm plasmids are extremely closely related; but the patterns obtained (particularly from PstI digests) enable the classification of the plasmids into groups. These bear a strong relation to time and place of isolation so that plasmids isolated on the same farm belong to the same group even when their host strains are of different antigenic types. It is concluded that these plasmids have evolved from a single plasmid.

Animals↗
PubMed1980

Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: homology with Tn3.

The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, gamma delta, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.

Base Sequence↗
PubMed1988

Structural instability of a bifunctional plasmid pZG1 and single-stranded DNA formation in Streptomyces.

Structural instability of a hybrid plasmid pZG1, consisting of Escherichia coli pBR322 and Streptomyces pIJ350 plasmids, has been studied in Streptomyces. After transformation of S. lividans 1326 and S. rimsus R6 protoplasts with pZG1, transformants harbored the intact pZG1 and various deleted plasmid forms. The pattern of deleted plasmids varied with the transformant colony age and changed upon subcultivation. The presence of intact pZG1 in at least a part of the Streptomyces colonies indicated that the plasmid was capable of replicating in the transformants and that deletion events occurred after at least one round of replication. Less instability of pZG1 in S. rimosus R6 was observed when this strain was transformed with the DNA isolated from the same strain. pZG1 and its various derivatives were found in S. lividans 1326 and S. rimosus R6 as double- and single-stranded DNA molecules. Structural instability of pZG1 could therefore be due at least in part to the presence of single-stranded DNA.

Chromosome Deletion↗