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Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 253 records · Page 14Linked to original sources
PubMed1983

Vasopressin and oxytocin mRNA regulation in the rat assessed by hybridization with synthetic oligonucleotides.

Vasopressin and oxytocin are nonapeptide hormones that regulate water metabolism and lactation, respectively. To study the regulation of the vasopressin and oxytocin genes at the mRNA level, we constructed a series of synthetic oligonucleotides, from 8 to 15 bases in length, for use in filter-blot hybridization assays (Northern blots) of hypothalamic mRNA levels and for primed synthesis of cDNAs from which we determined the nucleotide sequences of the 5' regions of the vasopressin and oxytocin mRNAs. A 20-fold increase occurred in the amounts of the two mRNAs present in the hypothalami of rats drinking 2% saline for three weeks. In addition, the sequence analyses of the cDNAs provided the complete amino acid sequences of the NH2-terminal signal peptides of the rat vasopressin and oxytocin precursors. Thus, synthetic oligonucleotides consisting of as few as eight nucleotides can be used to prime reverse transcription of specific cDNAs from hypothalamic RNA, and pentadecanucleotide hybridization probes readily detect changes in levels of vasopressin and oxytocin mRNAs in response to osmotic stress.

Amino Acid Sequence↗
PubMed1983

5'-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation.

Most eukaryotic mRNAs are characterized by the presence of a 5'-terminal cap structure (m7GpppN), and removal of the cap or translation of capped mRNAs in the presence of cap analogues (m7G) results in most cases in a significant decrease in the translational efficiency of the mRNAs. One way of explaining the importance of the 5'-cap is that cap-binding proteins recognize the cap structure, destabilize the mRNA secondary structure, and thus allow the 40S ribosomal subunit to bind to the mRNA [Sonenberg, N., Guertin, D., Cleveland, D., & Trachsel, H. (1981) Cell (Cambridge, Mass.) 27, 563-572]. Our data and those of others indicate that the translational efficiency of alfalfa mosaic virus RNA 4 (AMV-4 RNA), a naturally capped RNA, is not affected significantly by cap analogues or by removal of the cap. In order to examine the potential relationship between the function of the cap structure and secondary structure at the 5'-mRNA terminus, partial enzymatic digestion of capped AMV-4 RNA with single strand specific and double strand specific nucleases has been performed, and the experimental data have been compared with computer-generated models of AMV-4 secondary structure. In addition, the in vitro translatability of AMV-4 has been examined as a function of increasing potassium concentration, conditions that are likely to increase mRNA secondary structure. The nuclease-digestion results demonstrate that under native ionic conditions, the 5'-terminus of AMV-4 RNA is predominantly single stranded, although computer modeling and double-strand nuclease digestions indicate that the 5'-terminus can form weak base pairs with internal regions of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1983

Salt-induced conversion of B-DNA to Z-DNA inhibited by aflatoxin B1.

The carcinogen aflatoxin B1 was reacted with a polymer of alternating deoxyguanine and deoxycytosine residues to determine the effect that adduct formation has on the conversion of this polymer from the right-handed B-DNA form found at low salt concentrations to the left-handed Z-DNA form found at high salt concentrations. Reaction with aflatoxin strongly inhibited the salt-induced conversion of this polymer from B-DNA to Z-DNA. This inhibition could be detected even at relatively low binding levels.

Aflatoxin B1↗
PubMed1983

Formation of Z-DNA in negatively supercoiled plasmids is sensitive to small changes in salt concentration within the physiological range.

Negative supercoiling of the plasmid pBR322 with or without an insert of (dG-dC)n induces the formation of Z-DNA as measured by the binding of antibodies specific for Z-DNA. Increasing the concentration of Na+ (or K+) is shown to inhibit the B to Z-DNA conversion. This may be due to the effect of the cation on the B-Z junction. Using the data for B to Z-DNA conversion of the (dG-dC)n inserts, we have estimated the free energy change per base pair as well as the energy of the B-Z junction. In pBR322, a 14-bp segment [CACGGGTGCGCATG] is believed to form Z-DNA at bacterial negative superhelical densities under salt conditions which are similar to those found in vivo.

DNA↗
PubMed1983

Human parathyroid hormone gene (PTH) is on short arm of chromosome 11.

The human gene for parathyroid hormone (PTH) was chromosomally mapped using human-rodent hybrids and Southern filter hybridization of cell hybrid DNA. A recombinant DNA probe containing human PTH cDNA insert (pPTHm122) hybridized to a 3.7-kb fragment in human DNA cleaved with the restriction enzyme EcoRI. By correlating the presence of this fragment in somatic cell hybrid DNA with the human chromosomal content of the hybrid cells, the PTH gene was mapped to the short arm of the chromosome 11.

Animals↗
PubMed1983

Antibodies against Z DNA react with the macronucleus but not the micronucleus of the hypotrichous ciliate stylonychia mytilus.

Using indirect immunofluorescence, we studied the reaction of antibodies specific for left-handed Z DNA with the nuclei of the hypotrichous ciliate Stylonychia mytilus. In the vegetative cell, the macronucleus reacts strongly with these antibodies, but no reaction can be detected with micronuclei. However, an antibody that binds to denatured and right-handed B DNA reacts with both types of nuclei. No reaction of the anti-Z DNA antibody is seen in the macronuclear replication band. Digestion of macronuclei with DNAase I leads to a decrease in the anti-Z DNA antibody reaction. Some stages of the developing macronucleus were also investigated. No reaction is seen at the polytene chromosome stage, but following DNA elimination the nucleus is seen to react with the antibody.

Animals↗
PubMed1983

The sequence (dC-dA)n X (dG-dT)n forms left-handed Z-DNA in negatively supercoiled plasmids.

Z-DNA-specific antibodies have been used to demonstrate the formation of left-handed Z-DNA in sequences of (dC-dA)32 X (dG-dT)32 in negatively supercoiled plasmids. Z-DNA was found to form at physiological negative superhelical densities (sigma = -0.05) in the absence of high salt concentration or chemical modifications. The binding site of the antibody to the DNA sequences was demonstrated by crosslinking experiments. (dC-dA)n X (dG-dT)n sequences are widespread in eukaryotic DNA, and possible roles for such Z elements in chromatin activation or genome rearrangements are suggested.

Antibody Specificity↗
PubMed1983

Nucleotide sequence of the human parathyroid hormone gene.

From a lambda phage gene library we have isolated phage containing the gene encoding human preproparathyroid hormone. The phage were isolated by using both the plaque-hybridization technique and the in vivo recombination-selection technique. The human preproparathyroid hormone gene contains two intervening sequences that separate the gene into a 5' noncoding domain, a "prepro" sequence domain, and a domain containing the parathyroid hormone sequence and the 3' noncoding region. The gene is approximately 4,200 base pairs long. Restriction endonuclease analysis of human leukocyte DNA shows that the haploid human genome contains one copy of the preproparathyroid hormone gene. A 14-base-pair sequence of alternating purines and pyrimidines that has the potential of adopting the Z-DNA conformation lies 134 base pairs upstream from the presumed site of initiation of transcription.

Amino Acid Sequence↗
PubMed1983

Stabilization and detection of natural left-handed Z-DNA.

Left-handed Z-DNA is favored by nucleotide sequences with alternating purines and pyrimidines. The structural basis of this preference is described, as well as the factors which stabilize Z-DNA. The most important biological factors stabilizing Z-DNA are negative supercoiling and Z-DNA binding proteins. Experiments are described which make it possible to determine the nucleotide sequences of segments which form Z-DNA through the use of specific antibodies. Parts of the plasmid pBR322 form Z-DNA upon negative supercoiling. Similarly, three segments of the SV40 simian tumor virus form Z-DNA. They are located in the transcriptional enhancer region and may function, together with Z-DNA binding proteins, in facilitating the attachment of RNA polymerase to the DNA in chromatin. This is the first specific example suggesting a role of Z-DNA in regulating transcription.

Base Sequence↗
PubMed1983

Z-DNA and the polytene chromosome.

Indirect immunofluorescence shows that antibodies specific for the Z conformation of DNA bind to D. melanogaster polytene chromosomes in a specific and reproducible pattern. In our acetic-acid-fixed preparations, the binding is predominantly to interband regions, to puffs, and to some specific bands. As might be expected, the pattern of binding can be affected by cytological procedures. Bands and interbands show significant differences in their susceptibility to these procedures. Although further study is needed, our studies give no evidence that acetic acid treatment of intact cells is capable of causing conversion of B-form and Z-form DNAs. Instead, the experiments suggest that the Z conformation is preferentially available in interbands and a few other chromosome regions.

Animals↗
PubMed1983

Z-DNA-specific antibodies in human systemic lupus erythematosus.

Naturally occurring antibodies to left-handed Z-DNA have been shown to be present in the sera of human patients with systemic lupus erythematosus (SLE). These antibodies are of two types. One type reacts with both denatured DNA and Z-DNA. The other type is specific for Z-DNA and remained in the serum after removal of the cross-reactive antibody by extensive absorption on a denatured DNA affinity column. The antibodies appear to be specific for SLE and do not appear frequently in other rheumatic diseases. The presence of the antibody in SLE is correlated with the clinical manifestations of the disease, in parallel with antibodies to native and denatured DNA.

Antibody Specificity↗
PubMed1983

Molecular structure of d(m5CGTAm5CG): TA pairs in Z-DNA and the consequences.

The hexamer d(m5CGTAm5CG) crystallizes as a left-handed Z-DNA molecule in a isomorphous lattice to d(m5CGm5CGm5CG). The three-dimensional structure has been determined by x-ray diffraction and refined to a final R value of 16% at 1.2A resolution. The molecular structure is similar to the Z-DNA structure seen in d(m5CGm5CGm5CG) and d(CGCGCG). The presence of TpA sequence in the middle of the molecule has resulted in slight changes in the helical twist angles, base pair stacking and twist angles of the base pair planes. The hydration structure is significantly changed from both of methylated and non-methylated d(CGCGCG). A large hole which has no water molecule tightly bound to the DNA molecule is found inside of the deep groove of the DNA helix at the TpA sequence. This deterioration of the hydration structure is associated with destabilization of the Z-DNA structure. These characteristic features of the structure are compared with another Z-DNA hexamer contains TpA sequence, d(br5CGTAbr5CG).

Base Sequence↗
PubMed1982

Molecular structure of (m5 dC-dG)3: the role of the methyl group on 5-methyl cytosine in stabilizing Z-DNA.

The hexamer (m5 dC-dG)3 has been synthesized and its three-dimensional structure determined by a single crystal X-ray diffraction analysis. The structure has been refined to a final R value of 15.6% at 1.3 A resolution. The molecule forms a left-handed Z-DNA helix which is similar to the unmethylated Z-DNA structure. The presence of the methyl group has resulted in slight changes in the twist angle between successive base pairs and modification of some of the interatomic contacts. Methylation of cytosine in the C5 position is associated with a relative destabilization of the B-DNA structure and a stabilization through hydrophobic bonding of the Z-DNA structure.

5-Methylcytosine↗