Molecular weights,poly(A)-content,and partial separation of chick globin mRNAs

Poly(A)-containing 9S RNA from chick reticulocytes was electrophoresed on formamide-polyacrylamide gels. The molecular weight was determined to be 211 000±10 000 daltons. The RNA was separated into three different fractions with respect to molecular weight. These RNAs were translated in a wheat germ cell-free system. The lower molecular weight RNA directed up to 95% -chain synthesis, compared to 60% for the higher molecular weight RNA. This was accompanied by a relative increase for -chain synthesis with increasing molecular weight. It could also be shown by hybridization with labelled poly(U) that the average poly(A) length decreased from about 83 nucleotides for fraction I to 36 nucleotides for fraction III. Our results suggest that fractionation of avian 9 S globin mRNA by electrophoresis on formamide-polyacrylamide gels is dependent upon two parameters, namely differences in the lengths of the non-poly(A)-containing portion of the and mRNAs and differences in the poly(A) lengths.     

Effect of 7-methylguanosine-5′-phosphate on the translation of rat milk-protein mRNAs in the wheat-germ cell-free system

The translation of polyadenylated and of non-poly-adenylated RNA obtained from lactating rat mammary gland was almost totally inhibited by 0,5 mM 7-methylguanosine-5-phosphate in the wheat-germ cell-free system, This inhibition was maintained during the preparation of the 9S whey-protein mRNA and of the 12S and ISS casein mRNAs, Chemical decapping of these mRNAs caused a similar reduction of their activity . Although a large fraction of milk-protein mRNAs have been reported to lack 3-polyadenylation, these results show that the mRNAs in the mammary gland do contain a 5-terminal 7-methylguanosine cap.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Altering the 3′ UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3′-end maturation in vitro but not in vivo

The 3 ends of chloroplast mRNAs are produced by the processing of longer precursors. The 3 ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3-end-processing signals and RNA stability elements. In chloroplasts of the green alga Chlamydomonas reinhardtii, 3-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure. This cleavage is followed by exonucleolytic resection to generate the mature 3 end. In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3-end-processed products. However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3 end of the stable processing product differed from that of the wild-type product. To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3 UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3 end was generated. These results imply that Chlamydomonas atpB 3 processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.     

Characteristics of ribonucleic acids isolated from Botryodiplodia theobromae pycnidiospores

Nucleic acids isolated from dormant and germinated Botryodiplodia theobromae pycnidiospores contain five distinct species of RNA. They include two ribosomal species, two ribosomal-associated species and transfer RNAs. Sedimentation coefficients of 25.1S and 18S were obtained for the two ribosomal RNA species and 5.8S and 5S for the two ribosomal-associated RNA components. Molecular weights of 1.20, 0.67, 0.054 and 0.035x10⁶ daltons were obtained after formaldehyde treatment and electrophoresis on polyacrylamide gels for these same four RNAs. Methylated nucleotides were present in the transfer RNAs and large and small ribosomal RNAs; in contrast 5.8S and 5S RNAs contained few methylated nucleotides. In addition to the 5 distinct RNA species, polyadenylate-containing RNA was isolated from both dormant and germinated spores.Published with the approval of the Director as paper no. 5006, Journal Series, Nebraska Agricultural Experiment Station. The work was conducted under Nebraska Agricultural Experiment Station Project no. 21-17.     

Interactions of U1 RNP with heterogeneous nuclear RNA in rat Novikoff hepatoma nuclei

Summary The nature of the association of U1 RNA with rapidly sedimenting RNP structures in rat hepatoma nuclei was investigated. The effects of salt and proteinase K treatment on the stability of this bound form of U1 RNA were studied using sucrose density gradient analyses. Quantitation of the amount of U1 RNA remaining associated with large structures after treatment was used to assess the relative contribution of protein-protein(and protein-RNA) versus RNA-RNA interactions. Forty-eight percent of the total nuclear U1 RNA released by sonication was found in a bound form when the sonicate was centrifuged through gradients containing 50 mM NaCl. Fifty percent of this bound U1 RNA remained associated with rapidly sedimenting RNPs when the NaCl concentration was raised to 500 mM. To assess the contribution of protein independent interactions, large RNPs were completely deproteinized and their RNA moieties were then recentrifuged on gradients. By this analysis, 27% of the U1 RNA originally bound to hnRNPs was associated with rapidly sedimenting (>30 S) RNA (at 50 mM NaCl) suggesting their association by RNA-RNA hydrogen bonds. When the concentration of NaCl was 500 mM, 31% of the U1 RNA was associated with large RNA. Hence, approximately 30% of the U1 RNA molecules originally bound (or about 15% of the total nuclear U1 RNA) were found to be associated by RNA-RNA hydrogen bonds while the remainder of the binding of U1 RNP to hnRNP was by protein-protein and/or protein-RNA interactions.     

Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5-and 3 untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3 untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5 untranslated regions, but relatively little homology of the 5 untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3 untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.Abbreviations DM20 25 kDa proteolipid protein in myelin - PLP classic 30 kDa myelin proteolipid protein Special issue dedicated to Dr. Marjorie B. Lees.     

Effects of hemin on a lymphoblastoid cell line that expresses the human ɛ- and γ-globin genes

Summary Northern blot analysis using probes specific for each of the human embryonic (), fetal (), and adult () globin genes indicates that the human lymphoblastoid F-265 cells express the embryonic and fetal globin genes. Unlike: the erythroid cell line K562, in which globin RNA levels increase during treatment with hemin in culture, globin RNA levels decrease in F-265 cells in the presence of hemin. This effect is reversible after passage of F-265 cells in fresh medium without hemin. Both the rates of globin RNA synthesis and the presence of DNase I-hypersensitive sites in hemin treated and untreated F-265 cells were investigated to identify the levels at which globin gene expression is controlled.     

Different ζ globin gene deletions among Black Americans

Summary Four types of chromosomes with a deletion between the human embryonic and globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5 to the globin gene [(X⁺) or (X^-)]. The deletions are identifiable in Xba I digests of genomic DNA using an or a globin gene probe which yield fragments of 23 kb from (X⁺)–^* chromosomes or 27 kb from (X^–)–^* chromosomes. Digestion with other enzymes and probing with both and probes gave fragments typical of the two globin gene deletions previously identified in Polynesians. Among Black Americans, these globin gene deletions have been found in combination with globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult globin genes.     

Hemoglobin F production in heterocellular hereditary persistence of fetal hemoglobin and its linkage to the β globin gene complex

Summary Some types of nondeletional heterocellular hereditary persistence of fetal hemoglobin (HPFH) appear to be caused by mutations in the globin gene cluster near the globin genes, while in other cases the condition is associated with a gene or genes outside the globin gene complex. We have used DNA probes for chromosome 11 markers to localize the HPFH determinant in a large Australian kindred with nondeletional heterocellular HPFH. In 13 of the 58 family members studied the Hb F levels are increased to between 1.8% and 7.9%, the Hb F being composed predominantly of ^A chains. All family members were typed for restriction fragment length polymorphisms detected by probes from the globin gene complex, and the nearby genetic markers D11S12, INS, and HRAS. Linkage analysis showed HPFH is closely linked to the globin gene cluster (confidence limits of 0,0.0-0.19), D11S12 (0, 0.0-0.23) and the insulin gene (0,0.0-0.11). These data and the chain composition are consistent with HPFH in this family being caused by a mutation within the globin gene cluster.     

Homology of a 130-kb region enclosing the α-globin gene cluster,the α-locus controlling region,and two non-globin genes in human and mouse

The human -globin gene cluster (30 kb) is embedded in a GC-rich isochore very close to the telomere of Chromosome (Chr) 16p. The -Locus Controlling Region (-LCR) is located upstream of the adult -globin genes and has been shown to be essential for their expression. In this study we have been looking for expressed genes in the region upstream of the -globin cluster to understand the role of the LCR-like element in the expression and replication timing of flanking gene clusters. We show that the upstream -globin region is conserved over a 75-kb range and includes at least two oppositely transcribed non-globin genes, here referred to as Mid1 and Dist1. Complementary DNA sequences of 250 bp and 2.5 kb from Mid1 (coordinate-68) and Dist1 (coordinate-90 to-99), respectively, were isolated from human and mouse. The deduced partial amino acid sequences of these cDNAs are 81% and 95% identical for the Mid1 and Dist1 gene respectively. We have cloned a mouse cosmid contig which includes Dist1, Mid1, and the entire murine -globin cluster. The murine homolog of the -LCR was mapped upstream of the mouse globin genes at approximately the same position as in the human locus. Our results indicate that, in mouse and human, the -globin loci and their flanking sequences are homologous over a range of at least 130 kb. The structural homology of this region in both mammals suggests also a functional one and indicates the mouse as a potential model for studying the role of the -LCR controlling element in the regulation of expression and replication timing of the flanking gene clusters.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers M99623, M99624, M99625, and M99626.     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

6′-Mercaptoguanosine-resistance is related with purF gene encoding 5′-phosphoribosyl-1′-pyrophosphate amidotransferase in inosine-5′-monophosphate overproducing Brevibacterium ammoniagenes

From the gene library constructed with the chromosomal DNA of 6-mercaptoguanosine (MGS)-resistant strain Brevibacterium ammoniagenes IPR-1, a DNA fragment which conferred MGS-resistance to the wild-type strain B. ammoniagenes ATCC6872 was cloned. The purF gene encoding 5-phosphoribosyl-1-pyrophosphate amidotransferase was identified from this fragment and its nucleotide sequence was determined. Wild type purF gene was also cloned by polymerase chain reaction using chromosomal DNA of ATCC6872 as the template and its sequence was determined. Two nucleotides, 583 A and 1065 A, of MGS-resistant purF gene had been changed from 583 G and 1065 G by mutagenesis, respectively. Both changes at position 583 and 1065 were proved to be responsible for MGS-resistance by site-directed mutagenesis.     

Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes

Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17–20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as old mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of -globin in all systems, while the presence of the cap analogue m⁷G(5)p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5 terminal cap. Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.     

Comparison of human and chimpanzee ξ1 blobin genes

Summary The DNA base sequences of the entire chimpanzee 1 globin gene and an additional 1 kb of DNA flanking both the human and chimpanzee genes have been determined. Whereas the human 1 gene contains a termination codon in the sixth position, the chimpanzee gene appears to be functional. This finding confirms Proudfoot et al.'s suggestion that the human 1 gene was recently inactivated. Like the corresponding human 1 and 2 genes, the first and second introns of the chimpanzee 1 gene are occupied largely by tandem repeats of short oligonucleotides. These tandem repeats have undergone several rearrangements since the divergence of the human and chimpanzee 1 genes.     

Fetal hemoglobin variants in 80,000 Japanese neonates: high prevalence of Hb F Yamaguchi (AγT 80 Asp→Asn)

Summary A population-based screening of newborns for the structural variants of fetal hemoglobin was carried out in Osaka Prefecture, Japan, by isoelectric focusing of globin chains using dried blood on filter paper. Of 80,000 newborns, 18 had globin variants and 55 had globin variants. The incidence of globin variants (1/1,455) was much higher than that of globin variants (1/4,444). Structural studies were then carried out on the abnormal globins in 36 samples, and revealed that 25 of them were Hb F Yamaguchi (^AT 80 AspAsn). The prevalence of this variant in Japanese was estimated to be as high as one per 2,100.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum υ chain deduced from cDNA sequence

An RNA polymerase chain reaction strategy was used to amplify and clone a cDNA segment encoding for the complete constant part of the axolotl IgY heavy (C) chain. C is 433 amino acids long and organized into four domains (C1–C4); each has the typical internal disulfide bond and invariant tryptophane residues. Axolotl C is most closely related to Xenopus C (40% identical amino acid residues) and C1 shares 46.4% amino acid residues among these species. The presence of additional cysteines in C1 and C2 domains is consistent with an additional intra-domain S-S bond similar to that suggested for Xenopus C and C, and for the avian C and the human C. C4 ends with the Gly-Lys dipeptide characteristic of secreted mammalian C3, human C4, and avian and anuran C4, and contains the consensus [G/GT(AA)] nucleotide splice signal sequence for joining C4 to the transmembrane region. These results are consistent with the hypothesis of an ancestral structural relationship between amphibian, avian chains, and mammalian chains. However, these molecules have different biological properties: axolotl IgY is secretory Ig, anuran and avian IgY behave like mammalian IgG, and mammalian IgE is implicated in anaphylactic reactions.The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number X69492. Correspondence to: J. S. Fellah.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Exploration of RNA 3′ terminal locations in rat ribosome

The locations of the 3 ends of RNAs in rat ribosome were studied by a fluorescencelabeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3 ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe – fluorescein 5thiosemicarbazide (FTSC), indicating that the 3 termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3 terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3 end of 5 S RNA was located on the interface of two subunits. However, no fluorescencelabeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3 end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3 ends of ribosomal RNAs including oxidation with NaIO₄, reduction with KBH₄ and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3 ends of RNAs were not involved in the translation activity of ribosome.