Separation of nucleic acid precursors on an amphoteric surfactant modified monolith using combined eluent flow, pH and concentration gradient.

An RP monolithic column coated with an amphoteric carboxybetaine type surfactant has been used with a combined triple eluent concentration, pH and flow gradient ion chromatography technique for the simultaneous separation of up to 18 nucleotides, nucleosides and nucleobases. The separation of up to eight precursors on a 1 cm long monolithic microcolumn using the combined gradient approach is also shown. The method was applied to the separation of the above nucleic acid precursors in perchloric acid extracts of yeastolates samples.

Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction.

A procedure was developed for mapping origins of DNA replication in mammalian cell chromosomes based on determining the relative abundance of nascent DNA strands throughout a specific genomic region. The method entails purification of short strands of nascent DNA derived from recently activated origins and the quantification, within this sample, of the relative abundances of different adjacent DNA segments by a competitive polymerase chain reaction technique. It is expected that the abundance of defined markers within the origin region is greatest at the site where DNA replication begins. This origin mapping procedure (i) allows analysis of single-copy genomic regions, (ii) can be performed on cultured and primary cells in the absence of any chemical treatment, (iii) does not require cell synchronization, and (iv) allows mapping origins to within a few hundred base pairs. This high degree of resolution permits a study of the cis- and trans-acting elements required for origin function. Application of this method to single-copy sequences in mammalian cells has identified replication origins within an approximately 500-bp segment in the human lamin B2 gene domain and within an approximately 800-bp segment in the hamster dihydrofolate reductase gene locus.

A missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in Sephardic Jews.

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low density lipoprotein (LDL) receptor gene. Here, we characterize an LDL receptor mutation that is associated with a distinct haplotype and that causes FH in the Jewish Sephardic population originating from Safed, a town in northern Israel. The mutation was found in eight FH families originating from this community comprising 10% of heterozygote FH index cases screened in Israel. The mutation was not found in four additional FH heterozygotes whose hypercholesterolemia co-segregated with an identical LDL receptor gene haplotype. A guanine to cytosine substitution results in a missense mutation (asp147 to his) in the fourth repeat of the binding domain encoded by exon 4 of the LDL receptor gene. The mutant receptor protein was synthesized in cultured cells as a 120 kDa precursor form that failed to undergo normal processing to a mature cell surface form. Most of the receptor precursors were degraded in the endoplasmic reticulum. The small number of mutant receptors on the cell surface were unable to bind LDL or beta very low density lipoprotein. The abnormal behavior of the mutant receptor was reproduced by site-directed mutagenesis and expression of the mutant protein in CHO cells. The mutation can be diagnosed by allele-specific oligonucleotide hybridization of polymerase chain reaction amplified DNA from FH patients.

Identification of chicken calbindin D28K pre-messenger RNA sequences by polymerase chain reaction.

A transcribed RNA sequence encompassing the junction between the first intron and the second exon of the chicken calbindin D28K gene was copied in a cDNA fragment and subsequently amplified by polymerase chain reaction. When intestinal RNA is used as template, the appearance of the 161 bp amplified fragment is strictly dependent on the vitamin D status of the animal. In fact no amplified fragment is obtained when the RNA is extracted from the intestine of vitamin D-deficient chickens, while it is easily detected when the RNA is extracted only 30 min after injection with 1,25-dihydroxycholecalciferol. Conversely, the amplified fragment is obtained, irrespectively of the vitamin D status of the animal, when the RNA template is extracted from the brain. The appearance of unspliced RNA sequences upon vitamin D induction is followed, after a 30 min lag, by the appearance of the corresponding mature mRNA sequences.

Recognition of 5' and 3' splice site sequences in pre-mRNA studied with a filter binding technique.

A nuclear extract from HeLa cells was fractionated by DEAE-Sepharose chromatography. The obtained fractions were assayed for binding to an RNA transcript carrying a splice site sequence of 9-16 nucleotides by a filter binding technique. The U1 RNA-rich small nuclear ribonucleoprotein (snRNP) fractions, which showed binding activities for both 5' and 3' splice site RNAs, were studied for the sequence specificity of their binding. Results indicate that the U1-rich snRNP fraction can recognize both 5' and 3' splice site sequences. The U1 RNP, which was highly purified from the snRNP fractions, bound to at least some 5' splice site sequences, but not to a consensus 3' splice site sequence. Therefore, purified U1 RNP can directly recognize a 5' splice site, but not a 3' splice site. The binding activity for the 5' splice sites was lost either by digestion with micrococcal nuclease or by digestion of the 5' end of U1 RNA with RNase H and a complementary oligodeoxynucleotide, indicating the involvement of U1 RNA. Involvement of a protein moiety as well in this binding was suggested by the loss of binding activity upon heating at 60 degrees C. The binding activity to a 3' splice site sequence was not sensitive to digestion by micrococcal nuclease and was removed by protein A-coupled anti-Sm antibody. This activity was found in sucrose gradient fractions of about 8 S.

Multiple species of bovine adrenodoxin mRNA. Occurrence of two different mitochondrial precursor sequences associated with the same mature sequence.

Bovine adrenodoxin mRNA is found to consist of several distinct mRNA species which can be divided into two sets. Each set utilizes at least three of four separate poly(A)+ addition sites providing an explanation of the three sizes of adrenodoxin mRNA (1.75, 1.4, and 0.95 kilobases) previously observed in bovine adrenocortical RNA by this laboratory (Okamura, T., John, M.E., Zuber, M.X., Simpson, E.R., and Waterman, M.R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5705-5709). The two sets are distinguished from one another by a unique 5' sequence leading to two different amino acid sequences (approximately 10% homology) for the precursor portion of this nuclear encoded, mitochondrial protein. A common mature adrenodoxin sequence is encoded by both sets of mRNA. One set of RNAs is 10-fold more abundant than the other, but the levels of both sets can be induced by treatment of primary bovine adrenocortical cell cultures with adrenocorticotropin. The biological significance of these two types of adrenodoxin precursor sequences remains obscure.

Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 3' end of H3 messenger RNA.

Processing of the 3' end of sea urchin H3 histone pre-mRNA requires conserved sequence elements and the presence of U7 snRNA. A mutation in the conserved CAAGAAGA sequence of the H3 pre-mRNA that renders 3' processing of this precursor defective is shown to be suppressed by a compensatory change in the U7 snRNA, restoring the base-pairing potential of the two RNAs. RNA-RNA contacts between these two molecules appear to be an essential feature of the 3' processing reaction.

Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction.

Using S1 nuclease protection assays, we have examined the representation of cell cycle-dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase-free DNase I and subsequent high-salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by 1) DNA, RNA, and protein composition; 2) electrophoretic analysis of the proteins in each fraction; 3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and 4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.

Mechanism of recognition of the 5' splice site in self-splicing group I introns.

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.

Yeast class III gene transcription factors and homologous RNA polymerase III form ternary transcription complexes stable to disruption by N-lauroyl-sarcosine (sarcosyl).

Yeast Class III gene transcription factors and RNA polymerase III were used to form ternary transcription complexes on a tRNASer gene in vitro under UTP-limiting transcription conditions. These ternary transcription complexes were composed of template DNA, proteins, and RNA. We have shown that the RNAs contained in these complexes represented specifically initiated nascent pre-tRNASer transcripts. These nascent RNAs could be very efficiently elongated to full-length pre-tRNASer molecules, even in the presence of the ionic detergent sarcosyl. Partial purification (greater than 100-fold) of these sarcosyl-resistant ternary transcription complexes could be achieved in a single step via sucrose gradient sedimentation. Comparable sarcosyl-resistant ternary transcription complexes could not be formed using purified yeast RNA polymerase III as the only protein component of the complex.

Differential estrogen responsiveness of the vitellogenin and apo very low density lipoprotein II genes in the rooster liver.

The primary transcript of the chicken apo Very Low Density Lipoprotein II (apoVLDL-II) gene is formed almost immediately after a first estrogen administration, contrary to the appearance of the vitellogenin primary transcript which has a lag of at least 4 h. However, after a second estrogen administration the vitellogenin gene transcription shows no detectable lag (memory effect). After estrogen withdrawal, the primary transcripts of both genes rapidly decline to undetectably low levels. In the presence of estrogen, the half-lives of the mRNAs of apoVLDL-II and vitellogenin are 15 and at least 70 h, respectively, whereas in the absence of hormone they are only 3.5 and 5.5 h, respectively. Thus estrogen not only controls the transcription of both genes, but also the turnover of their mRNAs. In short, there appears to be a quantitative difference in the response of both genes.

Oligomerization of intervening sequence RNA molecules in the absence of proteins.

The intervening sequence RNA excised from the ribosomal RNA precursor of Tetrahymena forms linear and circular oligomers when exposed to a heating-cooling treatment in vitro. The reactions require no protein or external energy source. Oligomerization is different from other self-catalyzed reactions of the intervening sequence RNA in that it involves intermolecular rather than intramolecular recombination, producing RNA molecules that are substantially larger than the original. The observation that RNA molecules can catalyze their own oligomerization has possible implications for the evolution of chromosomes and for the replicative cycle of plant viroids and virus-associated RNA's.

Intron splicing: a conserved internal signal in introns of Drosophila pre-mRNAs.

The introns of Drosophila pre-mRNAs have been analysed for conserved internal sequence elements near the 3' intron boundary similar to the T-A-C-T-A-A-C in yeast introns and the C/T-T-A/G-A-C/T in introns of other organisms. Such conserved internal elements are the 3' splice signals recognized in intron splicing. In the lariat splicing mechanism, the G at the 5' end of an intron joins covalently to the last A of a 3' splice signal to form a branch point in a splicing intermediate. Analysis of 39 published sequences of Drosophila introns reveals that potential 3' splice signals with the consensus C/T-T-A/G-A-C/T are present in 18 cases. In 17 of the remaining cases signals are present which vary from this consensus just in the middle or last position. In Drosophila introns the 3' splice signal is usually located in a discrete region between 18 and 35 nucleotides upstream from the 3' splice point. We note that the Drosophila small nuclear U2-RNA has sequences complementary to C-T-G-A-T, one variant of the signal, and to C-A-G, one variant of the 3' terminus of an intron. We also note that the absence of any A-G between -3 and -19 from the 3' splice point may be an essential feature of a strong 3' boundary.

Purification and characterization of an endonuclease from Xenopus laevis ovaries which accurately processes the 3' terminus of human pre-tRNA-Met(i) (3' pre-tRNase).

We have previously reported that the primary transcript of the human tRNAMeti gene undergoes accurate processing to a mature 72-nucleotide species by activities present in the high speed supernatant of Xenopus laevis ovarian extracts (Zasloff, M., Santos, T., Romeo, P., and Rosenberg, M. (1982a) J. Biol. Chem. 257, 7857-7863). We now report the purification and characterization of the enzyme which processes the 3' terminus of the human pre-tRNAMeti species. The activity has been purified about 500-fold from a high speed supernatant of X. laevis ovarian extracts by standard methods. It appears to function as a single polypeptide with a molecular weight of about 97,400. The enzyme generates the mature 3' terminus with a single endonucleolytic cut, also yielding the intact 3' trailer. The endonuclease has a striking preference for the 5' processed pre-tRNAMeti, exhibiting little or no activity in vitro on the intact primary transcript. The enzyme acts similarly with the pre-tRNAAla species of Bombyx mori, suggesting that it possesses a broad substrate range. The requirement of the 3' processing endonuclease for a processed 5' terminus suggests that eukaryotic pre-tRNA processing should follow an ordered cutting sequence in vivo with processing of the 5' leader preceding 3' end maturation.

Detection of beta-globin mRNA precursor in heterogeneous nuclear ribonucleoprotein associated with U1-RNP by using anti-RNP antibody.

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) fractions were isolated from Friend erythroleukemia cells and separated by 15-45% sucrose gradient centrifugation. The distribution of small nuclear RNAs (snRNAs) in hnRNP fractions indicated that the snRNAs are associated with hnRNP particles. HnRNP fractions were incubated with normal IgG or anti-U1 RNP IgG, and the resulting immunocomplexes were isolated by binding to a protein A-Sepharose column. HnRNP was found in bound fractions only when anti-U1 RNP IgG was used. By Northern hybridization of RNA extracted from the immunocomplexes with a beta-globin genomic DNA probe, 15S beta-globin mRNA precursors and 10S mature mRNA were detected. These findings suggest the existence of a complex of U1 RNP particles and hnRNP particles containing beta-globin pre-mRNA.

[Hybridization of low molecular weight nuclear RNA with pre-mRNA from erythroid bone marrow cells and rabbit mRNA]. / Gibridizatsiia nizkomolekuliarnykh iadernykh RNK s pre-mRNK éritroidnykh kletok kostnogo mozga i mRNK globina krolika.

Hybridization of labeled low molecular weight (LMW) nuclear RNA's to pre-mRNA from rabbit non-matured erythroid bone marrow cells or globin mRNA from reticulocytes revealed three RNA species having approximately 90, 100 and 160 nucleotides which are were specifically hybridized with purified cytoplasmic globin messenger RNA, while one (100 nucleotides) was also hybridized with rabbit 18S rRNA. The identity of these rabbit RNAs to LMW RNAs described for other animal species, as well as their possible hybridization sites and function are discussed.

Hemagglutination assay of polypeptide coded by the pre-S region of hepatitis B virus DNA with monoclonal antibody: correlation of pre-S polypeptide with the receptor for polymerized human serum albumin in serums containing hepatitis B antigens.

The receptor for polymerized human as well as chimpanzee serum albumins has been identified on the 55-amino acid polypeptide coded by the pre-S region of hepatitis B virus DNA. Monoclonal antibodies were raised against a synthetic polypeptide of 19 amino acid residues representing a hydrophilic region of the pre-S amino acid sequence deduced from hepatitis B virus DNA. Sheep erythrocytes fixed with glutaraldehyde were coated with monoclonal antibody against the synthetic polypeptide to develop a hemagglutination assay for pre-S polypeptide. The pre-S polypeptide was detected in the serum containing hepatitis B surface antigen particles along with hepatitis B e antigen, with titers in parallel with those of the receptor for polymerized human serum albumin.

RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.

To determine whether a specific nucleotide sequence is required to direct polyadenylation of a simian virus 40 early pre-mRNA in a soluble HeLa whole-cell lysate, we constructed a series of rearranged and deleted DNA templates, transcribed them in vitro, and determined whether the resultant RNAs could be polyadenylated when incubated in whole-cell lysate. When a 237-base-pair DNA fragment encoding the 3' end of the simian virus 40 early pre-mRNA was transferred to recombinant plasmids encoding RNAs that were not substrates for polyadenylation, the resultant RNAs could now be polyadenylated efficiently. In one case, the chimeric RNA was polyadenylated even more efficiently than was the original simian virus 40 early transcript. Analysis of the RNAs produced from the deletion mutant templates revealed that only RNAs containing at least one copy of the AAUAAA sequence situated near the 3' end and implicated in 3'-end formation and polyadenylation in vivo could be polyadenylated in vitro. Surprisingly, this sequence directed polyadenylation of pre-mRNAs not only when near the RNA 3' end, i.e., 50 nucleotides or less away, but also when the 3' end was situated over 400 nucleotides downstream. Thus, our results show that a polyadenylic acid polymerase activity in HeLa lysates can recognize a specific nucleotide sequence in pre-mRNA and then, in the absence of the nucleolytic cleavage that presumably occurs in vivo, locate the RNA 3' end and use it as a primer for polyadenylic acid synthesis.

Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.

The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage-ligation reactions that result in pre-rRNA splicing and IVS cyclization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic comparison. Thus, IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.