Physical mapping of the transfer RNA genes on lambda-h80dglytsu+36

The three Escherichia coli transfer RNA genes of the DNA of the transducing phage λ80cI857S^?t68dglyTsu⁺₃₆tyrTthrT (abbreviated λh80T), which specify the structures of tRNA^Gly₂(su⁺₃₆), tRNA^Tyr₂ and tRNA^Thr₃, have been mapped by hybridizing ferritin-labeled E. coli tRNA to heteroduplexes of λh80T DNA with the DNA of the parental phage (λh80cI857S^?t68) and examining the product in the electron microscope. The DNA of λh80T contains a piece of bacterial DNA of length 0·43 λ unit³ that replaces a piece of phage DNA of length 0·46 λ unit, proceeding left from B · P′ (the junction of bacterial DNA and phage DNA) (i.e. att⁸⁰). A cluster of three ferritin binding sites, and thus of tRNA genes, is seen at a position of 0·24 λ unit (1·1 × 10⁴ nucleotides) to the left of B· P′. The three tRNA genes of the cluster are separated by the unequal spacings of 260 (±30) and 140 (± 30) nucleotides, proceeding left from B·P′. The specific map positions have been identified by hybridization competition between ferritin-labeled whole E. coli tRNA with unlabeled purified tRNA^Tyr₂ and with unlabeled partially purified tRNA^Gly₂. The central gene of the cluster is tRNA^Tyr₂. The tRNA^Gly₂gene is probably the one furthest from B·P′. Thus, the gene order and spacings, proceeding left from B·P′, are: tRNA^Thr₃, 260 nucleotides, tRNA^Try₂, 140 nucleotides, tRNA^Gly₂.     

Specific binding of bovine immunoglobulins to Sepharose and Sephadex

Bacteriophage T5 BglII/HindIII DNA fragment (803 basepairs), containing the genes for 2 tRNAs and 2 RNAs with unknown functions, was cloned in the plasmid pBR322. The analysis of DNA sequence indicates that tRNA genes code isoacceptor tRNAs^Ser (tRNA^Ser₁ and tRNA^Ser₂) with anticodons UGA and GGA, respectively. The main unusual structural feature of these tRNAs is the presence of extra non-basepaired nucleotides in the joinings of stem ‘b’ with stems ‘a’ and ‘c’.     

Total nucleotide sequence of a nearly full-size DNA copy of satellite tobacco necrosis virus RNA

pSTNV-1 is a chimera plasmid that contains a nearly full-size double-stranded DNA copy of the satellite tobacco necrosis virus RNA genome (see preceding paper by van Emmelo et al., 1980) and we report here the complete nucleotide sequence of this STNV² DNA insert. The results show that except for 23 nucleotide pairs corresponding to the 5′ end of STNV RNA, a full-size STNV DNA copy is present in pSTNV-1. The total nucleotide sequence of the STNV genome contains 1239 residues. The amino acid sequence of the coat protein can be deduced from the 5′ half of the DNA message strand and shows a rather hydrophobic carboxyl-terminal region and a basic amino-terminal region. The 3′ untranslated part of the viral RNA is 622 nucleotides long. A secondary structure model for the 5′ end showing an interaction with a segment in the 3′ half is proposed. The 3′ end region can be folded into a transfer RNA cloverleaf-like structure with an anticodon for AUG.     

Identification and nucleotide sequence of the sup8-e UGA-suppressor leucine tRNA from Schizosaccharomyces pombe.

Summary Using the translation of rabbit globin mRNA in wheat germ extracts as an assay for ochre and opal suppression, a UGA suppressor tRNA from Schizosaccharomyces pombe strain sup8-e was purified by column chromatography and two-dimensional gel electrophoresis. The purified tRNA can be aminoacylated with leucine by a crude aminoacyl-tRNA synthetase preparation from a wild type S. pombe strain, and has high activity in the suppressor assay. By a combination of post-labeling fingerprinting and rapid gel sequencing methods the nucleotide sequence of this suppressor tRNA was determined to be: pG-C-G-G-C-U-A-U-G-C-C-ac⁴C-G-A-G-D-G-D-G-D-A-A-G-G-G-m ₂ ² G-G-C-A-G-A--U-U^*-C-A-m¹G-C-C-C-U-G-C-U-G-U-U-G-U-A-A-A-A-C-G-m⁵C-G-A-G-A-G-T--C-G-m¹A-A-C-C-U-C-U-C-U-G-G-C-C-G-C-A-C-C-A_OH. The anticodon sequence U^*CA is complementary to the UGA codon. An interesting feature of the suppressor tRNA is an expanded anticodon loop of nine nucleotides owing to an A-C nonpair at the first anticodon stem position.     

The effect of growth temperatures on the in vivo ribose methylation of Bacillus stearothermophilus transfer RNA

When Bacillus stearothermophilus was cultured at 70 and at 50 °C, 1.4 times as many methyl groups were incorporated into tRNA produced at the higher temperature compared to that produced at the lower. This was due predominantly to a threefold increase in the 2′-O-methylribose moieties of the tRNA. The type and quantity of the base methylated nucleotides in the tRNAs produced in cultures grown at 70 and 50 °C were almost identical. The base methylated nucleotides found were: m²Ap, ms²Ap, ms²i⁶Ap, an unidentified i⁶Ap derivative, m⁶Ap, m₂⁶Ap, m¹Gp, m⁷Gp, m⁵Up-(Tp), and an unidentified methylated Up or Cp.³ The nucleotide m⁷Ap, never before reported to be a constituent of tRNA, has been tentatively identified as a component of B. stearothermophilus tRNA.     

Complete nucleotide sequence of the leader rna synthesized in vitro by vesicular stomatitis virus

The complete nucleotide sequence of the leader RNA synthesized in vitro by the Indiana serotype of vesicular stomatitis virus is presented. The sequence was determined by the technique described by Donis-Keller, Maxam and Gilbert (1977) in combination with the standard two-dimensional fingerprint techniques described by Barrell (1971). The leader RNA contains 48 nucleotides variably terminating at the 3′ terminus with cytosine (68%) and adenosine at position 47 (32%). Since the leader RNA is complementary to the 3′ terminal portion of the viral genome RNA, the first 48 nucleotides from the 3′ end of the genome RNA can be deduced. The leader RNA contains repetitive and palindromic sequences with a polypurine sequence at its 3′ terminus. The possible role of some of the sequences is discussed.     

Nucleotide recognition seouence at the cleavage site of Haemophilus aegyptius II (Hae II) restriction endonuclease

We have determined the nucleotide sequence recognized by the restriction endonuclease Hae II from Haemophilus aegyptius which cleaves the simian virus 40 (SV40) DNA at a single specific site. By using terminal radioactive labeling of the cleavage site at both the 5′ and 3′-ends we have deduced the recognition sequence,

with elements of a two-fold rotational symmetry. The endonuclease produces staggered ends with protruding 3′-terminated single-strands, four nucleotides in length. In plasmid RSF 2124 DNA, which contains multiple Hae II cleavage sites, it was observed that the 5th nucleotide from the 3′ terminus is either a pdA or a pdG, indicating alternating recognition sequences.     

A yeast arginine specific tRNA is a remnant aspartate acceptor

High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNA^Asp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA₄^Arg. This tRNA has a sequence strikingly similar to that of tRNA^Asp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA₄^Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA₄^Arg into an aspartate acceptor, as efficient as tRNA^Asp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA₄^Arg. The latent aspartate acceptor capacity in a contemporary tRNA^Arg leads to the proposal of an evolutionary link between tRNA₄^Arg and tRNA^Asp genes.     

The nucleotide sequence of rat liver tRNAAsn

The major species of asparagine specific tRNA was isolated from rat liver, degraded to oligonucleotides, and shown to have the nucleotide sequence pG-U-C-U-C-U-G-U-m¹G-m²G-C-G-C- A-A-D-C-G-G-D-X-A-G-C-G-C-m²G-ψ-ψ-C-G-G-C-U-Q-U-U-t⁶A-A-C-C-G- A-A-A-G-m⁷G-D-U-G-G-U-G-G-Z-ψ-C-G-m¹A-G-C-C-C-A-C-C-C-A-G-G-G- A-C-G-C-C-A_OH. Although this tRNA contains several modified nucleotides in their expected positions, it is unique in having X, 3-(3-Amino-3-carboxy-n-propyl)uridine in loop I rather than in loop III; Q, 7-(4,5-cis-dihydroxyl-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine in the wobble position of loop II; and Z, an unknown, and presently uncharacterized nucleoside, at position 23 from the 3′ terminus usually occupied by ribothymidine.     

Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs

U₁ snRNP² isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U₁ molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U₁ core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle.When a less drastically purified U₁ snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected.When a mixture of U₁, U₂, U₄, U₅ and U₆ snRNPs, which contains the same four/five polypeptides as U₁ snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U₂, U₄ and U₅ were found in addition to those derived from U₁. No fragment derived from U₆ was found.In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U)_nG with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U₂, where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U₆ snRNA, it does not contain the sequence A(U)_nG which correlates well with the fact that no U₆ snRNA fragment seems to resist micrococcal nuclease digestion.     

The nucleotide sequence of 4.5 S rRNA from tomato chloroplasts

The nucleotide sequence of 4.5 S rRNA from tomato (Lycopersicum esculentum Mill) chloroplasts has been determined to be:

. The 4.5 S rRNA is 103 nucleotides long and has a free 5′-terminal hydroxyl group. It shows at high degree of homology with chloroplast 4.5 S rRNA from other plants.     

Preliminary x-ray diffraction studies on lobster ATP: L-arginine phosphotransferase.

An endonuelease R.HindIII, prepared from Hemophilus influenzae strain Rd, degrades foreign DNA, but not homologous DNA. Phage T7 DNA is also resistant to the enzyme. Fragments of phage λ DNA produced by treatment with R.HindIII have been labelled at their 5′ termini and analysis of the radioactive nucleotides in pancreatic DNAase digests of these fragments revealed a single 5′ terminal sequence. From this and other data we conclude that the enzyme recognizes and cleaves DNA at the following nucleotide sequence,

giving termini bearing short cohesive ends.     

The anticodon of the maize chloroplast gene for tRNA Leu UAA is split by a large intron.

The maize chloroplast gene encoding tRNA Leu UAA has been sequenced. It contains a 458 base pair intron between the first and second bases of the anticodon. The tRNA is 88 nucleotides long (the 3'-terminal CCA sequence included which, however, is not encoded by the gene) and differs in only four nucleotides (modified nucleotides are not considered) from the corresponding isoacceptor from bean chloroplasts. The unusual position of the intron in this maize chloroplast tRNA gene suggests a splicing model different from that generally accepted for eukaryotic split tRNA genes.     

Polymorphisms in the 5′-UTR of <Emphasis Type="Italic">PTEN</Emphasis> and other gene polymorphisms in normal Japanese individuals

Polymorphisms are distributed differently in populations, including those of regions, ethnic groups, and diseased patients. In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced the 5′-untranslated region (5′-UTR) of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene and the gene promoter, intron, and exon nucleotides of p53, p14 ^ARF , murine double minute 2 (MDM2), and the β₂- and β₃-adrenoceptor (?AR). We found polymorphisms in these regions, including a deletion at positions ?465 to ?463 and a substitution at position ?404 in PTEN and a substitution at position ?4924 in p14 ^ARF , in normal individuals. Individuals with or without the PTEN polymorphism harbored a different distribution of polymorphisms, including simultaneous alterations in nucleotides of p53, MDM2, and β₃-AR, and also harbored some polymorphic nucleotides located in the same set of associatively altered nucleotides. Our results show that multiple nucleotides, including the PTEN nucleotides, are altered in normal Japanese individuals and provide useful information for genotyping studies in individuals and populations.     

On the Concentration Gradient across a Spherical Source Washed by Slow Flow

A model has been numerically analyzed to help interpret the orienting effects of flow upon cells. The model is a sphere steadily and uniformly emitting a diffusible stuff into a medium otherwise free of it and moving past with Stokes flow. Its properties depend primarily upon the Peclet number, Pe, equal to a · v_∞/D, i.e., the sphere's radius, a, times the free stream speed, v_∞, over the stuff's diffusion constant, D. As Pe rises, and washing becomes more effective, the average surface concentration, _{s a} falls (Figs. 2 and 5) and the residual material becomes relatively concentrated on the sphere's lee pole (Figs. 2 and 4). Specifically, as Pe rises from 0.1 to 1, the relative concentration gradient, G, rises from 0.7 to 5.0 per cent and to the point where it is rising at about 8 per cent per decade; by Pe 1000, G = 22.1 per cent. From Pe 1 through 1000, G/(1 - _{s a}), or the gradient per concentration deficiency remains at about 26 per cent suggesting that G approaches a ceiling of about 26 per cent. Also from Pe 1 through 1000, the average mass transfer co-efficient nearly equals that previously calculated for spheres maintaining constant surface concentration instead of flux. The complete differential equation without approximations, the Gauss-Seidel method, and an approximation for the outer boundary condition were used.     

Origin of splice junction phosphate in tRNAs processed by HeLa cell extract

Two cloned tRNA genes that contain intervening sequences, yeast tRNA_UCG^Ser and Xenopus laevis tRNA^Tyr, were transcribed in HeLa cell extract. Precursor tRNAs were formed, and were converted to spliced products by a process of excision-ligation. The novel sequences resulting from ligation of tRNA half-molecules were examined by fingerprinting and nearest neighbor analyses. The results indicate that during tRNA splicing in HeLa cell extract, the 3′-terminal phosphate of the 5′ half-molecule is incorporated into a normal 3′,5′-phosphodiester linkage that forms the splice junction. This ligation pathway in HeLa cell extract is distinct from the one described previously in wheat germ extract, which involves formation of 2′-phosphomonoester, 3′,5′-phosphodiester

linkage with the 3′,5′-bond derived from a 5′-terminal phosphate.     

Primary structure of E. coli alanine transfer RNA: relation to the yeast phenylalanyl tRNA synthetase recognition site

Nucleotide sequence comparison of tRNAs aminoacylated by yeast phenylalanyl tRNA synthetase (PRS) have lead to the proposal that the specific nucleotides of the dihydrouridine (diHU) stem region and adenosine at the fourth position from the 3′ end are involved in the PRS recognition site. Kinetic analysis and enzymatic methylation have shown that the size of the diHU loop and the methylation of guanine at position 10 from the 5′ end both directly affect the PRS aminoacylation kinetics. $\text{E. coli}$ tRNA₁^A1a, which is aminoacylated by PRS, should therefore have 1- the specific nucleotides of the diHU stem region and, 2- adenosine at position 4 from the 3′ end. The PRS aminoacylation kinetics of this tRNA indicates that this molecule 3- has a diHU loop of 8 nucleotides and 4- has an unmethylated guanine at position 10 from the 5′ end. We report here the complete sequence of $\text{E. coli}$ tRNA₁^A1a and confirmation of each of these four predictions.