End Nucleotide Research Articles

Expression and chromosomal localization of the gene for the human transcriptional repressor GCF.

GCF is a human transcriptional regulator that represses transcription of certain genes and is encoded by a 3-kilobase (kb) mRNA (Kageyama, R., and Pastan, I. (1989) Cell 59, 815-825). The expression of GCF was examined in a variety of clonal cell lines. The 3.0-kb GCF mRNA was found to be expressed at the highest level in HUT 102 cells (derived from a T-cell lymphoma). Elevated levels of the GCF mRNA were also noted in KATO III and AGS (gastric carcinomas), FEM-X (melanoma), and U266B1 (myeloma) cell lines. A human fibroblast cell line (WI38) did not express GCF mRNA, and no cross-hybridization to a mouse cell line (NIH 3T3) or monkey cell line (CV-1) could be detected. The GCF cDNA also hybridizes to RNA species of 4.5 and 1.2 kb. The 4.5-kb RNA has the same general expression pattern as the GCF mRNA. Hybridization of cellular RNA with various probes derived from the 3-kb cDNA revealed that the 4.5-kb RNA species only hybridizes to GCF cDNA probes from the extreme 5' end. By using single-stranded RNA probes, hybridization to the three RNA species was detected with the antisense probe for the 5' end (nucleotides 1-561). The single-stranded antisense probe for the region encompassing nucleotides 561-1692 hybridized to the 3.0- and 1.2-kb RNA species. The sense probes for these regions did not hybridize to these RNAs. The GCF gene was localized to a single locus, the chromosome 2 p11.1-11.2 region, by in situ hybridization. Treatment of human KB epidermoid carcinoma cells with phorbol 12-myristate 13-acetate (PMA) lead to a rapid induction of GCF RNA after 1 h and a decline to lower than control levels after 6 h. Epidermal growth factor receptor mRNAs were not increased by PMA until 2 h after treatment and were at their highest level only after GCF mRNAs were decreased. The 4.5- and 1.2-kb RNAs were also induced by PMA with the same kinetics as the GCF mRNA. These results show that the GCF gene is widely expressed in human tissues and cell lines and that the 4.5- and 1.2-kb RNAs have similar expression patterns.

Both yeast W double-stranded RNA and its single-stranded form 20S RNA are linear.

Most yeast strains carry a cytoplasmic double-stranded RNA (dsRNA) molecule called W, of 2.5 kb in size. We have cloned and sequenced most of W genome (1), and we proposed that W (+) strands were identical to 20S RNA, a single-stranded RNA (ssRNA) species, whose copy number is highly induced under stress conditions. Recently it was proposed that 20S RNA was circular (2). In this paper, however, we demonstrate that both W dsRNA and 20S RNA are linear. Linearity of W dsRNA is shown by the stoichiometric labelling of both strands of W with 32P-pCp and T4 RNA ligase. The last 3' end nucleotide of both strands is about 70 to 80% C and 20 to 30% A. Linearity of 20S RNA is directly demonstrated by a site-specific cleavage of 20S RNA with RNase H, using an oligodeoxynucleotide complementary to an internal site of 20S RNA. The cleavage produced not one but two RNA fragments expected from the linearity of 20S RNA.

Tissue-specific expression of two gamma-glutamyl transpeptidase mRNAs with alternative 5' ends encoded by a single copy gene in the rat.

Two different cDNAs have been isolated and characterized from a rat kidney cDNA library. The two cDNA sequences are identical in the coding region and in the 144 bases upstream from the initiation codon but have alternate sequences (154 and 138 bases) at their 5' ends. Primer extension analysis on kidney mRNA reveals that both cDNAs are full-length and correspond to two mRNAs of nearly the same size (2142 and 2127 bases). Synthesis of two mRNAs with alternative 5' ends can be explained only by initiation at two separate promoters on the single rat gamma-glutamyl transpeptidase (GGT) gene. The alternate 5' end nucleotide sequences were used as probes to detect the corresponding mRNAs in several rat tissues. In the kidney, the expression of both RNAs was detected by in situ hybridization in the distal part of the proximal convolutions of the renal tubule. Northern blot analysis of kidney mRNAs reveals that the expression of both mRNAs increases from birth to the adult stage. Neither of these two transcripts is expressed in the liver or in seminal vesicles in which a larger mRNA (2.4 kilobase pairs) is transcribed from the same gene. Thus, two GGT mRNAs, initiated on two separate promoters on the single GGT gene, are expressed in the rat in a tissue-specific manner and coordinately regulated.

Sardinian Gγ-HPFH: A T → C Substitution in a Conserved “Octamer” Sequence in the Gγ-Globin Promoter

A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3' end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

Sardinian G gamma-HPFH: a T----C substitution in a conserved "octamer" sequence in the G gamma-globin promoter

A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3′ end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

Cloning and 5' end nucleotide sequences of two juvenile hormone-inducible vitellogenin genes of the African migratory locust.

Screening of lambda libraries prepared with locust (Locusta migratoria) genomic DNA yielded overlapping clones containing two complete vitellogenin genes, designated A and B, along with extensive 3'- and 5'-flanking sequences. Genes A and B are about 12 kb and 10.5 kb long, respectively, and both hybridize on Northern blots with 6300-nucleotide RNAs previously identified as vitellogenin mRNAs. Each gene contains a large intron near the 5' end and must also contain other introns. Repetitive "Lm1 elements," similar to the human Alu repetitive DNA elements, and other repeated sequences occur within the large intron and in the 3'- and 5'-flanking regions of each gene. Southern blots showed no cross-hybridization between the major portions of mRNA-coding sequences of genes A and B. DNA sequences have been determined for the 5' terminal exons and flanking regions of both genes. Both genes contain short (56-58 nucleotide) 5' leader exons, which, unlike the rest of the coding region, are highly conserved in sequence (87% identical) and code for amino acids that resemble the amino-terminal sequences of vertebrate vitellogenins. The upstream 5'-flanking sequences of the two genes contain several short stretches of similarity that may be involved in juvenile hormone regulated expression.

Existence of single-strand interruptions in the genomic DNA of mycobacteriophage I3

The genomic DNA of mycobacteriophage I3 harbours single-strand interruptions and is susceptible to degradation by nuclease S1, leading to random fragmentation. The native DNA serves as an efficient template for DNA polymerase I showing the presence of free 3′-OH termini which provide the priming points within the genome. The single-strand regions show no specificity for the 5′-end nucleotides flanking these interruptions and all 4 nucleotides are present with a probability equivalent to their percentage in the total DNA. The single-strand interruptions are an intrinsic character of this genome and are present irrespective of the host on which the phage is propagated.

Examination of prebiotic adaptor models

Some models of the prebiotic protein synthesizing mechanism involve hairpin helices as adaptor molecules (Hopfield, 1978: Kuhn and Waser, 1981: Crothers, 1982). We examined the feasibility of hairpin strands to act as primitive adaptor molecules (Kuhn and Waser, 1981). Both right and left handed helices were used in the analysis of the stereochemical feasibility of the simultaneous recognition of the anticodon by the codons on messenger RNA and peptide condensation between two amino acids carried by adjacent hairpin strands. Initially right handed hairpin helices were constructed using the anticodon loop of the crystal structure of tRNA (Sussman et al., 1978). A complex of two such hairpin strands aligned on a messenger RNA was constructed, such that the anticodons recognize the codons by the normal Watson-Crick base pairing scheme. The dihedral angles in the junction region of the nucleotide chain between the two codons were varied to bring the two hairpin strands together for peptide bond condensation. The conformation corresponding to the closest approach of the two hairpin helices corresponds to a distance between the 2'hydroxyl oxygens on the 3'-end nucleotides in the two hairpins, (where the amino acid gets attached) of 26.5A. It is evident that if two amino acids are attached to the 2' oxygens of the adjacent hairpin helices, peptide bond condensation between the two amino acids is not possible. In the second instance, we examined the feasibility of hairpin strands with left handed helices making use of the crystal structure of Z-DNA (Wang et al., 1979). Kuhn and his co-workers in a series of papers (Kuhn and Waser, 1981: Lehmann and Kuhn, 1984) suggest a sequence of three (anticodon) nucleotides to be in the loop region of the hairpin helices. Gralla and Crothers (1973) suggest that a hairpin loop of three nucleotides is destabilizing thermodynamically. They further propose that a loop of three nucleotides will exist when the bases flanking the loop are not in a base pairing configuration. Marky and Olson (1982) suggest that even a loop length of four will be possible only when there is a lot of flexibility in the chain. From energy minimization studies of dApdApdAp, Ponnuswamy and

Accumulation and specific cleavage of 5S RNA in the isthmus of laying hen oviduct. Evidence for three chicken 5S RNA

RNA extracts from the isthmus of laying hen oviduct contain truncated 5S RNA molecules that were found to be shorter at their 5' terminus as compared to native 5S RNA I and II. Moreover one of the truncated species differs from 5S RNA I by the absence of the 3' end nucleotide. The truncated forms increase of about 70% the total 5S RNA (intact + truncated) in the isthmus, as compared to the other studied tissues. Furthermore 5S RNA I is heterogeneous: 25% have A instead of U at the 3' end, and some evidence was obtained for the existence of two 5S RNA I conformers.

Molecular cloning of Lassa virus RNA: Nucleotide sequence and expression of the nucleocapsid protein gene

Lassa virus RNA isolated from purified virus particles was polyadenylated and reverse transcripts were cloned into the PstI site of plasmid pUC9. Clones containing sequences of the smaller (S) segment of the Lassa virus genome were identified by hybridization with purified S RNA. They were characterized by their ability to hybridize with fragments of 3′-labeled Lassa virus S RNA and with each other, and by restriction mapping. The largest insert was 1830 by long and began with the 3′-terminal 19-base sequence characteristic of all arenavirus S RNAs so far analyzed. The virus complementary strand contained a single large open reading frame, beginning at the ATG nearest its 5′ end (nucleotides 103–105) and terminating with a TGA triplet at position 1813–1815, that encodes a protein of 570 amino acids. A recombinant was constructed which expressed the gene as a fusion protein in Escherichia coli. The product was a 60-kDa polypeptide which reacted with monoclonal antibodies specific for the nucleocapsid protein. Comparison of the predicted amino acid sequence with the corresponding sequences deduced from the nucleotide sequences of the other arenavirus S RNAs, lymphocytic choriomeningitis (LCM) and Pichinde, reveals a considerable degree of similarity between Old and New World arenaviruses.

Isolation and the 5'-end nucleotide sequence of Bacillus licheniformis alpha-amylase gene.

We have isolated and determined the 5'-end nucleotide sequence of the alpha-amylase gene from Bacillus licheniformis ATCC 14580. The alpha-amylase produced by this strain is thermostable and of liquefying type. The gene was originally cloned in a bacteriophage lambda 1059 vector. A subclone containing a 5.3 X 10(3)-base insert in pBR322 was further characterized. The nucleotide sequence coding for the 5' end of the structural gene together with the sequence coding for the upstream control regions was determined. The deduced N-terminal amino acid sequence was identical with the previously published amino acid sequence of B. licheniformis alpha-amylase. There was also very strong homology to the N-terminal sequence of Bacillus amyloliquefaciens alpha-amylase. The Mr of the thermostable alpha-amylase, as determined in vitro in a cell-free transcription/translation system of Escherichia coli, was about 55 000.

Complete nucleotide sequence of the mRNA coding for the N protein of vesicular stomatitis virus (New Jersey serotype)

The nucleotide sequence of the mRNA encoding the nucleocapsid protein of the New Jersey serotype (Ogden strain) of vesicular stomatitis virus (VSV) was determined from two overlapping cDNA clones spanning almost entirely the coding region of the mRNA. The 5′-terminal noncoding sequence present in the mRNA but not in the cDNA clones was determined from a primer extended to the 5′ terminus of the mRNA. The mRNA is 1329 nucleotides long (excluding polyadenylic acid) and encodes a protein of 422 amino acids. The nucleotide sequence was compared with the previously determined nucleotide sequence of the nucleocapsid protein of the Indiana serotype. An overall identity of 67.7% was found between the two serotypes. The only place where insertions and/or deletions have occurred during the evolution of the two viral genes from their presumed common ancestor is in the untranslated region. The nonidentical nucleotides are distributed throughout the length of the mRNA although not in an entirely random manner. The predicted amino acid sequence demonstrates that both proteins are initiated from the initiator codon located at the same distance from the 5′ end (nucleotides 14 to 16) and contain the same number of amino acids. An overall identity of more than 80% of the amino acid sequence was observed between the two proteins when conservative replacements of amino acids were considered.

Studying Poliovirus with Infectious Cloned cDNA

The application of molecular cloning to the study of poliovirus is discussed. Initially cDNA clones representing the entire genome of poliovirus type 1 (Mahoney) were prepared. These molecular clones were used for determining the complete nucleotide sequence of the viral genome. Subsequently a single cDNA clone representing the entire poliovirus genome was constructed. When this cDNA clone was transfected into cultured mammalian cells, infectious poliovirus was produced. The transfection-derived poliovirus was identical to wild-type virus by several criteria, including RNase T1-oligonucleotide fingerprint and 5' end nucleotide sequence. The availability of infectious cloned cDNA should make possible new approaches to the study of poliovirus.

Sequence analysis and precise mapping of the 3′ ends of HeLa cell mitochondrial ribosomal RNAs

The 3′-terminal segments of the two HeLa cell mitochondrial ribosomal RNA species have been sequenced by subjecting 3′-end-labeled samples of highly purified components to RNAase T 1 or RNAase A digestion, fingerprinting the products and further characterizing them by secondary enzymatic digestion. The results, when correlated with the mitochondrial DNA sequence, clearly indicate that the 3′ end nucleotide of the transcribed moiety of almost 90% of the 12 S rRNA molecules corresponds in the DNA sequence to a nucleotide immediately contiguous to the following tRNA Val gene, while the transcribed moiety of the remainder of the 12 S rRNA molecules is shorter by one, or perhaps two or three, nucleotides. Both the major and the minor 12 S rRNA species have been found to be, in the majority, oligoadenylated at their 3′ ends, exhibiting mostly stretches of one to five A residues. A similar analysis of the 16 S rRNA has shown the occurrence of two major types of transcribed moiety comprising about 90% of the total, and several minor ones. Some ambiguities in the precise localization in the mitochondrial DNA sequence of the 3′-terminal residues of the 16 S rRNA transcripts have been resolved by high resolution mapping experiments utilizing the S 1 protection technique and analysis of the products on sequencing gels. The results clearly show that the 3′ termini of the two major types of transcribed moiety correspond in the DNA sequence to the nucleotide immediately adjacent to the tRNA Leu gene or to the preceding one; furthermore, the 3′ ends of the minor types of transcribed moiety form with those of the major types a cluster of nucleotides, mostly contiguous, in the mitochondrial DNA sequence. All species of transcribed moiety have been found to be oligoadenylated. The present results point to a certain imprecision in the process leading to the formation of the 3′ ends of 16 S rRNA and, together with other data, support a model whereby termination of transcription, rather than processing, is responsible for generating these 3′ ends. The observation of oligoadenylation in the HeLa cell mitochondrial rRNAs confirms and extends similar findings in the homologous hamster RNAs, and suggests a general role of adenylation in the processing or termination of mitochondrial DNA transcripts.

Further characterization of mRNA's of mouse hepatitis virus: presence of common 5'-end nucleotides.

The mouse hepatitis virus strain A59 codes for seven RNA species in the infected cells. These virus-specific RNAs were found to be polysome associated and therefore likely to represent mRNA's. All of them have common 3'-end sequences (Lai et al., J. Virol. 39:823-834, 1981). Their structure was further studied with respect to their 5'-end sequences. It was found that all of these mRNA's contained cap structures at their 5' ends. Furthermore, the cap-containing oligonucleotides which represent the sequences immediately adjacent to the 5' ends were found to be the same for most, if not all, of the seven virus-specific mRNA's. These sequences are also identical to the 5'-end sequences of the virion RNA genome. The 5'-end sequences were tentatively determined to be 5'-cap-N-UAAG. The presence of the common nucleotides in all of the virus-specific RNAs in mouse hepatitis virus strain A59 suggests several possible mechanisms of synthesis for these RNAs. The significance of these findings is discussed.

Origin and characterization of short DNA chains in Escherichia coli.

One of the major problems in the study of DNA replications involves the presence of numerous short, non-nascent DNA chains in the cell. Such chains often contaminate nascent DNA preparations, making accurate analysis of the replicative DNA difficult. This complication can be avoided by the use of hydroxyapatite chromatography. Previous results from this laboratory had shown that short nascent DNA chains could be eluted from hydroxyapatite at low phosphate concentrations because of their non-covalent association with protein. Additional data now indicate that this isolation procedure yields a DNA preparation which contains only nascent DNA and is essentially free of non-nascent chains. 5'-Terminal labelling patterns and molecular weight distributions show that only nascent DNA chains from the DNA-protein complex and can therefore be separated from non-nascent chains if the isolation is performed under non-denaturing conditions. The protein involved in the complex appears to be rather specific for DNA chains in the replication fork since short chains resulting from the excision of dUMP immediately after replication also form the DNA-protein complex. Using this isolation technique, the 5'-terminal nucleotides of nascent DNA chains were determined in an effort to learn something about the chain initiation process. DNA was pulse-labeled in several ways, purified by hydroxyapatite chromatography, and labeled at the 5' end with [32P]phosphate. Two-dimensional thin-layer chromatography of the 32P-labeled nucleotides, obtained after enzymatic hydrolysis, revealed that all four deoxyribonucleotides are present at the 5' end in approximately equal amounts. In most cases, this 5'-terminal nucleotide distribution does not vary significantly, even when the conditions of pulse-labeling are changed. Only if the cells are allowed to run out of thymine is a variation in the relative amounts of 5'-end nucleotides detected. In this case, the amount of dTMP at the 5' end decreases significantly, reflecting the low thymine levels and indicating that the cell can compensate for this deficiency by utilizing other available nucleotides. From these results, it appears that DNA chain initiation is essentially random with respect to the nucleotide used in the initiation process.

Radioimmune assays and molecular studies that place Anopheles B and Turlock serogroup viruses in the Bunyavirus genus (Bunyaviridae).

Molecular analyses indicate that Turlock virus (TUR, Turlock serogroup) and Boraceia virus (BOR, Anopheles B serogroup) have virion RNA species and polypeptides comparable in size to those of members of the Bunyavirus genus and unlike those of members of the newly defined Phlebovirus, Nairovirus, or Uukuvirus genera (Bunyaviridae). The 11 terminal 3' end nucleotides of the three virion RNA species of both BOR and TUR viruses (HOUCAUCACAUG...) are identical in sequence to the 3' end sequences of the viral RNA species of snowshoe hare (SSH) and La Crosse bunyaviruses (LAC, California serogroup, Bunyavirus genus). Competition radioimmune assays (RIA), using iodinated LAC nucleocapsid polypeptide (N), or LAC glycoproteins (G1, G2), and LAC rabbit hyperimmune antisera, or iodinated Oriboca (ORI, Group C, Bunyavirus genus) N, or G1 and G2 polypeptides and LAC antisera, or iodinated Bunyamwera (BUN, Bunyamwera serogroup, Bunyavirus genus) N, or G1 and G2 polypeptides and BUN or LAC antisera, have indicated that the virion polypeptides of BOR virus share antigenic determinants with these other bunyaviruses. Competition RIA analyses also have shown that TUR virus shares antigenic determinants with LAC virus. The competition RIA analyses have confirmed the antigenic relationships of LAC, SSH, trivittatus, Bwamba, Aino, Simbu, Mermet, Guaroa, Lumbo, Tahyna, ORI, Anopheles A, BUN, Capim, Guama and Shark river viruses (Bunyavirus genus members), and lack of antigenic relationships between Karimabad, or Chagres, or sandfly fever, Sicilian, Viruses (Phlebovirus genus members), and the bunyaviruses, LAC, ORI, or BUN.

Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands.

Human mitochondrial DNA contains two physically separate and distinct origins of DNA replication. The initiation of each strand (heavy and light) occurs at a unique site and elongation proceeds unidirectionally. Animal mitochondrial DNA is novel in that short nascent strands are maintained at one origin (D-loop) in a significant percentage of the molecules. In the case of human mitochondrial DNA, there are three distinct D-loop heavy strands differing in length at the 5' end. We report here the localization of the 5' ends of nascent daughter heavy strands originating from the D-loop region. Analyses of the map positions of 5' ends relative to known restriction endonuclease cleavage sites and 5' end nucleotides indicate that the points of initiation of D-loop synthesis and actual daughter strands are the same. In contrast, the second origin is located two-thirds of the way around the genome where light strand synthesis is presumably initiated on a single-stranded template. Mapping of 5' ends of daughter light strands at this origin relative to known restriction endonuclease cleavage sites reveals two distinct points of initiation separated by 37 nucleotides. This origin is in the same relative genomic position and shows a high degree of DNA sequence homology to that of mouse mitochondrial DNA. In both cases, the DNA region within and immediately flanking the origin of DNA replication contains five tightly clustered tRNA genes. A major portion of the pronounced DNA template secondary structure at this origin includes the known tDNA sequences.

TRNA punctuation model of RNA processing in human mitochondria

A 3'-end proximal segment of most of the putative mRNAs encoded in the heavy strand of HeLa cell mtDNA has been partially sequences and aligned with the DNA sequence. In all cases, the 3'-end nucleotide of the individual mRNA coding sequences has been found to be immediately contiguous to a tRNA gene or another mRNA coding sequence. These and previous results indicate that the heavy (H) strand sequences coding for the rRNA, poly(A)-containing RNA and tRNA species form a continuum extending over almost the entire length of this strand. We propose that the H strand is transcribed into a single polycistronic RNA molecule, which is processed later into mature species by precise endonucleolytic cleavages which occur, in most cases, immediately before and after a tRNA sequence.

Purification of an endonuclease present in Chrysaora quinquecirrha venom.

AbstractA DNase was isolated from the nematocyst venom of the sea nettle (Chrysaora quinquecirrha). This enzyme was purified 880-fold by sequential affinity chromatography. The enzyme had a pH optimum of 6.8, a molecular weight of 110,000 daltons, an isoelectric point of 6.9, and was active only on single-stranded DNA. The activity of the DNase could be inhibited by bivalent ions (Ca2+, Mg2+, and Zn2+) but was stimulated by sodium chloride. The nettle DNase could incise superhelical DNA only in low saline concentrations. Nettle DNase lacked 3′- to 5′-exonucleolytic activity, and its endonucleolytic activity produces cleaved reaction products containing 5′-phosphoryl or 3′-hydroxyl end nucleotides.