The 3' terminal sequence of chicken ovalbumin messenger RNA and its comparison with other messenger RNA molecules.

The subcellular distribution of actin in embryonic chick fibroblasts and brain was examined biochemically. Several gentle extraction procedures, which did not cause the breakdown of muscle filamentous actin, caused the release of large amounts of “cytoplasmic actin” in a monomeric form. This did not behave as a precursor or degradation product of filamentous actin in pulse label experiments and failed to form filaments under the same conditions as muscle actin. However, when it was purified and concentrated it was able to form aggregates which were very similar to paracrystals of muscle filamentous actin. These results suggest that cytoplasmic actin is at a higher concentration than muscle actin before it will polymerize, and that in the cell much of it is either monomeric or in a labile state.     

3' non-translated region of human alpha-globin messenger RNA does not affect translation efficiency

The relative translation efficiency of three synthetic alpha-globin mRNAs differing by their 3' non-translated end was measured in vitro in a rabbit reticulocyte lysate. Results showed that substituting the 3' non-translated end of human alpha 2 globin mRNA by the 3' non-translated end of chimpanzee alpha 1 or alpha 2 mRNAs has no effect on translation efficiency. In contrast, the introduction of the alpha-Quong-Sze mutation (alpha 125, Leu----Pro) in human alpha 2 mRNA led to a 50% apparent reduction in globin synthesis due to the instability of the alpha-Quong-Sze globin chain. We conclude that human alpha 1 and alpha 2 globin mRNAs have the same translation efficiency, and that the reduction, previously reported, in the kinetics of alpha-globin synthesis by alpha 2 mRNA carrying the alpha-Quong-Sze mutation is due to the instability of the alpha-Quong-Sze globin chain only.     

Nucleotide sequence of bovine prolactin messenger RNA. Evidence for sequence polymorphism

Hybrid molecules containing DNA sequences complementary to bovine pituitary mRNA were constructed in the Pst I site of pBR322 by the dC . dG tailing technique. Recombinant plasmids containing bovine prolactin (bPRL) sequences were amplified in bacteria and identified by hybridization to purified [32P]bPRL cDNA sequences. Nucleotide sequence analysis was performed on the inserts from two of the positive clones. One clone, pBPRL72, contained a 982-base pair insert that included 67 nucleotides of the 5'-untranslated region, the complete coding region of the preprolactin protein (690 nucleotides), and the entire 3'-untranslated region (150 nucleotides) of bPRL mRNA. The nucleotide sequence analysis of clone pBPRL72 predicted the sequence of a 30-amino acid signal peptide and confirmed the published amino acid sequence of the protein with one exception. A comparison of the pBPRL72 cDNA sequence with a second bPRL clone, pBPRL4, revealed four silent nucleotide differences. Three of the base changes occurred in the third position of amino acid codons, and one occurred in the 3'-noncoding region. The sequence polymorphism suggests the existence of alleles or multiple loci for bPRL that do not alter the protein structure.     

Nucleotide sequence of rat liver aldolase B messenger RNA

The nucleotide sequence of messenger RNA encoding rat liver aldolase B has been determined by sequence analysis using recombinant cDNAs cloned in bacterial plasmids. The sequence contains part of the 5'-untranslatable region (68 nucleotides), the entire coding region (1092 nucleotides), and the complete 3'-untranslatable region (387 nucleotides), excluding the poly(A) tail. A potential ribosomal-binding site is located about 30 nucleotides upstream from the initiation codon. The amino acid sequence of rat liver aldolase B is composed of 364 amino acids and has 70% homology with rabbit muscle aldolase A.     

Secondary structure at the 3' terminal region of RNA coliphages: comparison with tRNA

Secondary structure models for the 3' non-coding region of the four groups of coliphage RNA are proposed based on comparative sequence analysis and on previously published data on the sensitivity of nucleotides in MS2 RNA to chemical modification and enzymes. We report the following observations. (1) In contrast to the coding regions, the structure at the 3' terminus is characterized by stable regular helices. We note the occurrence of the loop sequences 5'-GUUCGC and 5'-CGAAAG, that are reported to confer exceptional stability to stem structures. These features are probably present to promote the segregation of mother and daughter strands during replication. (2) Comparison of homologous helices indicates that only those base pair substitutions are allowed that maintain the thermodynamic stability. (3) We have compared the structure of phage RNA with tRNA. Overall similarity is low, but one common element may exist. It is a quasi-continuous helix of 12 basepairs that could be the equivalent of the 12 basepair long coaxially stacked helix, formed by the T psi C arm and the aminoacyl acceptor arm in tRNA. As in tRNA, this structure element starts after the fourth nucleotide from the 3' end. (4) Phage RNA contains a large variable region of about 35 nucleotides bulging out from the quasi-continuous helix. We speculate that the variable loop in present-day tRNA could be the remnant of the variable region found in phage RNA. The variable region contains overlapping binding sites for the replicase enzyme and the maturation protein. This common binding site may serve as a switch from replication to packaging.     

Nucleotide sequence of a cDNA clone for human aldolase: a messenger RNA in the liver

Nearly complete cDNA clones for human aldolase A mRNA were isolated from human liver cDNA library and the nucleotide sequence determined. Using the cDNA clone as a probe the length of human aldolase A mRNAs, isolated from the skeletal muscle, liver and placenta tissues, was measured by RNA blotting and estimated to be 1,600 nucleotides for skeletal muscle mRNA and 1,700 nucleotides for both the liver and placenta mRNAs, indicating that different species of mRNA coding for human aldolase A were expressed in the different tissues.     

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.     

Nucleotide sequence at the 5'' end of ovalbumin messenger RNA from chicken.

DNA-sequence analysis of 300 nucleotides from the region of cloned, double-stranded ovalbumin cDNA corresponding to the 5' end of ovalbumin messenger RNA was accomplished using the technique of Maxam and Gilbert (Proc. Nat. Acad. Sci. USA (1977) 74,560-564). The AUG initiation codon was located 52 nucleotides from the AT linkers used in cloning and immediately adjacent to the amino terminal peptide of ovalbumin, indicating the absence of a "signal peptide" in this protein. The nucleotide sequence coding for a phosphorylated peptide from ovalbumin was also found. These results demonstrate that the coding portion of mRNAov begins near the 5' end of the molecule leaving some 600 nucleotides of noncoding information at the 3' end.     

Nucleotide sequence of nucleolar U3B RNA.

U3A, U3B, and U3C are three distinct molecular weight nucleolar RNAs present in Novikoff hepatoma ascites cells. The primary nucleotide sequence of U3B, the most prominent of these U3 species, was determined. Purified U3B RNA was subjected to various enzymatic digestion procedures, including digests of 32P-labeled U3B RNA, RNA ligase, and polynucleotide kinase labeling, for determination of its primary sequence which is: (formula: see text). The 5'-terminus of the RNA has a "cap" and localized purine-rich regions were found near the 3'-terminus, which have been incorporated into a hydrogen-bonded region in a proposed secondary structure of the molecule.     

Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA.

By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.     

Nucleotide sequence analysis of DNA. XI. The 3' terminal sequences of bacteriophage lambda and phi 80 DNA

Two procedures have been developed and applied to the determination of the 3′ terminal sequences of λ DNA and φ80 DNA. In the first procedure, each 3′ terminus was specifically labeled with a single ³²P-nucleotide. Radioactive oligonucleotides of different lengths were obtained by partial pancreatic deoxyribonuclease digestion. From the characteristic mobilities of these oligonucleotides in two dimensional fractionation systems, the 3′ terminal sequence -ACCCGCG for the r-strand and -GGTTACG for the l-strand of λ DNA have been determined. In the second procedure, approximately six nucleotides were removed from each 3′ terminus with exonuclease III, and they were replaced with radioactive nucleotides by partial repair synthesis. After enzymatic digestion and sequence analysis, the above sequences have been confirmed. The 3′ terminal sequences in φ80 DNA are identical to those in λ DNA at least up to the fifth nucleotide from the 3′ ends.     

A novel progesterone-induced messenger RNA in rabbit and human endometria. Cloning and sequence analysis of the complementary DNA

Complementary DNAs (cDNAs) prepared from messenger RNAs (mRNAs) isolated from endometria of 5 day pregnant rabbits were inserted into the plasmid pBR322. A library of 2400 recombinant plasmid clones was prepared and screened by differential in situ hybridization with cDNAs prepared from mRNAs of rabbits either injected with progesterone or untreated by the hormone. Clones encoding uteroglobin were identified and discarded. Several progesterone-induced and progesterone-repressed clones were identified. One of them corresponded to a relatively frequent mRNA (0.2% of clones in the library) of 2300 nucleotides. The induction of this messenger RNA by progesterone was totally suppressed by the antagonist RU486. This compound displayed a limited agonistic activity when administered alone. A very small increase in mRNA concentration was observed after estradiol administration. The messenger RNA was also found in the liver (where it was constitutively expressed), the ovaries, and the Fallopian tubes of rabbits. A cross-hybridizing messenger RNA was detected in human endometrium during the luteal phase. Sequence analysis showed that the messenger RNA encoded a protein of 370 amino acids with a calculated molecular weight of 40,800. A search in Genbank and National Biomedical Research Foundation data banks showed no identity or marked similarity with previously published DNA or protein sequences.