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D L Hatfield

Publications and source records attributed to D L Hatfield.

At least 55 records · Page 3Linked to original sources
PubMed1994

Reconstitution of the biosynthetic pathway of selenocysteine tRNAs in Xenopus oocytes.

Selenocysteine is cotranslationally introduced into a growing polypeptide in response to certain UGA codons in selenoprotein mRNAs. The biosynthesis of this amino acid initiates by aminoacylation of specific tRNAs (designated tRNA([Ser]Sec)) with serine and subsequent conversion of the serine moiety to selenocysteine. The resulting selenocysteyl-tRNA then donates selenocysteine to protein. In most higher vertebrate cells and tissues examined, multiple selenocysteine isoacceptors have been described. Two of these have been determined to differ by only a single modified residue in the wobble position of the anticodon. In addition, the steady-state levels and relative distributions of these isoacceptors have been shown to be influenced by the presence of selenium. In order to gain a better understanding of the relationship between these tRNAs and how they are regulated, both the Xenopus selenocysteine tRNA gene and an in vitro synthesized RNA have each been injected into Xenopus oocytes and their maturation analyzed. In this system, selenium enhanced RNA stability and altered the distribution of isoacceptors that differ by a single ribose methylation. Interestingly, the biosynthesis of one of these modified nucleosides (5-methylcarboxymethyl-2'-O-methyluridine), which has been identified only in the wobble position of selenocysteine tRNA, also occurs in oocytes. Examination of the modified residues in both the naturally occurring Xenopus selenocysteine tRNA and the products generated from exogenous templates in oocytes demonstrated the faithful reconstruction of the biosynthetic pathway for these tRNAs.

Animals↗
PubMed1994

Selenocysteine tRNA and serine tRNA are aminoacylated by the same synthetase, but may manifest different identities with respect to the long extra arm.

Selenocysteine (Sec) tRNA([Ser])Sec donates Sec to protein, but interestingly, this amino acid is synthesized on tRNA which is first aminoacylated with serine. Thus, the identity elements in tRNA([Ser])Sec for aminoacylation correspond to elements for seryl-tRNA synthetase recognition. As tRNA([Ser])Sec has low homology to the tRNA(Ser) isoacceptors, it would seem then that the identity elements in tRNA([Ser])Sec involve (1) very specific sequences, (2) conformational features, and/or (3) different points or domains for tRNA[Ser]Sec:synthetase and tRNASer:synthetase recognition. Initially, we confirmed that the same synthetase aminoacylates both tRNAs by showing that a mutant tRNA[Ser]Sec which has a blocked 3'-terminus is a competitive inhibitor of tRNASer aminoacylation with a partially purified and a highly purified seryl-tRNA synthetase preparation. The discriminator base (base G73) is essential for aminoacylation of tRNA([Ser])Sec and tRNA(Ser), while the long extra arm plays an important role which seems to be orientation- and length-specific in tRNA(Ser) and, in addition, may manifest sequence specificity in tRNA([Ser])Sec. This difference in the tRNA recognition specificity is discussed. The acceptor stem, DHU stem, and T phi C stem contribute to the recognition process, but to a lesser extent than the discriminator base and the long extra arm.

Animals↗
PubMed1993

Mapping of nucleic acid binding in proteolytic domains of HIV-1 reverse transcriptase.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane and renatured after SDS-PAGE; the proteins are then probed with the primer analogues 32P-labeled d(T)16 or 32P-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 approximately equal to 300. By contrast, a C-terminal domain fragment termed p30(303 approximately equal to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Dietary selenium affects methylation of the wobble nucleoside in the anticodon of selenocysteine tRNA([Ser]Sec).

We reported previously that the presence of selenium in culture media of mammalian cells influences both the steady-state levels and distributions of two tRNA isoacceptors involved in the insertion of selenocysteine into protein in response to certain UGA codons. In this study, we demonstrate an increase in the levels of these isoacceptors in rats fed a selenium-adequate diet compared to animals fed a selenium-deficient diet, as well as a shift in the relative distribution toward the tRNA which elutes later from an RPC-5 column. These effects were found to occur in a tissue-specific manner. Both selenocysteine tRNAs were isolated from rat liver, sequenced, analyzed by mass spectrometry, and shown to differ only by ribose 2'-O-methylation of 5-methylcarboxymethyluridine that occurs in the wobble position of the anticodon. This modified nucleoside has been documented previously only in yeast tRNA while the corresponding 2'-O-methylribose derivative has not been observed. The structure of these nucleosides was established by mass spectrometry and confirmed by chemical synthesis. Although the role of methylation of the wobble nucleotide is not known, the differences in elution properties from RPC-5 columns are consistent with other experimental observations indicating that a change in tRNA conformation accompanies this methylation.

Animals↗
PubMed1991

Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding.

Properties of primer recognition by purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66 homodimer have been investigated. Earlier studies had shown that RNA-directed DNA synthesis catalyzed by HIV-1 RT proceeds by an ordered mechanism in which template-primer combines with the free enzyme to form the first complex in the reaction scheme, and it was also shown that primer alone is a competitive inhibitor of template-primer. In this study, enzyme-primer binding has been further characterized utilizing pd(T)8 and pd(T)16 as model primers and UV cross-linking to covalently trap the enzyme-primer complexes. Competition experiments with several authentic primers, including tRNA(3Lys), indicate that pd(T)n binds to the kinetically significant primer binding site of RT. Salt reversal experiments suggested that the free energy of pd(T)n binding to RT has a large nonelectrostatic component. Binding of pd(T)n to p66-RT is not affected by dNTPs and does not require the presence of template. The site of UV cross-linking of pd(T)16 was localized to the NH2-terminal half of p66 by use of V8 protease hydrolysis and microsequencing. Our results indicate that a polynucleotide binding site is in close proximity to residues in the peptide comprising amino acids 195 approximately 300. This region could be either a single-stranded template or single-stranded primer binding site; however, we have documented the specificity of binding with oligonucleotides that act as primer in the in vitro DNA synthesis reaction. Therefore, this d(T)16 binding site may be part of a primer-binding groove within the HIV-1 reverse transcriptase.

Animals↗
PubMed1991

Selenocysteyl-tRNA occurs in the diatom Thalassiosira and in the ciliate Tetrahymena.

Selenocysteyl-tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl-tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl-tRNA suggests that selenocysteine-containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.

Animals↗
PubMed1989

Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA.

The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%).

Animals↗
PubMed1989

Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA.

An in vitro system to assay translational readthrough of the UAG termination codon at the murine leukemia virus (MuLV) gag-pol junction was developed by using rabbit reticulocyte lysates programmed by SP6-generated Moloney MuLV gag-pol mRNA. Under conditions in which the suppressor activity of the lysate was dependent on addition of tRNA, it could be shown that readthrough synthesis was stimulated to approximately the same extent by equivalent amounts of tRNA from MuLV-infected and uninfected NIH 3T3 cells. Analysis of glutamine tRNA, which mediates suppression in vivo, showed that the level of glutamine acceptor activity and the chromatographic profile of glutamine isoacceptors were unchanged following virus infection. On the basis of these results, we conclude that the suppressor tRNA occurs normally within the tRNA population of uninfected cells and need not be induced in response to virus infection.

Animals↗
PubMed1989

Suppression of UAA and UGA termination codons in mutant murine leukemia viruses.

Genomes of mammalian type C retroviruses contain a UAG termination codon between the gag and pol coding regions. The pol region is expressed in the form of a gag-pol fusion protein following readthrough suppression of the UAG codon. We have used oligonucleotide-directed mutagenesis to change the UAG in Moloney murine leukemia virus to UAA or UGA. These alternate termination codons were also suppressed, both in infected cells and in reticulocyte lysates. Thus, the signal or context inducing suppression of UAG in wild-type Moloney murine leukemia virus is also effective with UAA and UGA. Further, mammalian cells and cell extracts contain tRNAs capable of translating UAA and UGA as amino acids. To our knowledge, this is the first example of natural suppression of UAA in higher eucaryotes.

Animals↗
PubMed1986

Effects of post-transcriptional base modifications on the site-specific function of transfer RNA in eukaryote translation.

The site-specific function in translation of several naturally occurring mammalian transfer RNAs has been studied in a series of investigations with some similarities to studies in other laboratories of tRNAs in suppression. Equal amounts of aminoacyl-tRNA isoacceptors with contrasting isotopes were added in pairs to reticulocyte lysates and allowed to incorporate their amino acids into rabbit globin. Rates of incorporation from unlimiting amounts of each isoacceptor into the corresponding amino-acid-containing sites were determined. The tRNAs of each isoacceptor pair differed as to post-transcriptional base modifications. The natural occurrence of these isoacceptors can be correlated with rates of cellular division, with more rapidly dividing and neoplastic cells containing hypomodified tRNA. The overall incorporation of lysine into globin from a fully modified tRNALys that decodes AAG is faster by 25 to 30% than from the corresponding hypomodified tRNALys. There is considerable scatter in values for incorporation ratios at different lysine-containing sites, with the hypomodified isoacceptor even being preferred at one site. The AAG decoding isoacceptors are capable of translating AAA although much more slowly than AAG. In translating AAA, in contrast to translating AAG, the hypomodified tRNALys isoacceptor is preferred. A Y base-deficient hypomodified tRNAPhe isoacceptor found only in some kinds of rapidly dividing tumor cells donates its phenylalanine preferentially to globin in competition with the fully modified Y-containing tRNAPhe of liver by 15 to 17%. There is a considerable range of incorporation ratios at the different phenylalanine-containing sites of the globin subunits. No correlation can be made between the isoacceptor preferred and the phenylalanine codon being translated. The incorporation of histidine from a fully modified tRNAHis-containing Q base in its anticodon, compared with that from the hypomodified counterpart isoacceptor that lacks Q base and that occurs in rapidly dividing cells, showed no difference in their ability to incorporate overall or into individual histidine-containing sites. There is little evidence that adjacent bases or codons in messenger RNA affect the tRNAs preferred in the translation of most sites. A striking pattern of tRNA preference was observed in three cases in which there are tandem codons, with the same codon appearing twice in succession.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Prolactin homogeneously induces the tRNA population of mouse mammary explants.

Explants of mouse mammary glands were cultured with and without prolactin in the presence of inorganic [32P] to estimate the effect of prolactin on tRNA synthesis. Labeled tRNA was extracted and characterized by two-dimensional gel electrophoresis. tRNA synthesis was 2-3 fold greater in the presence of prolactin; and the synthesis rate of each resolvable tRNA species was increased proportionally. tRNA populations from mouse mammary tissues at three stages of development were also examined. Alterations were noted between early pregnant and fully lactating tissues. The results of this study provide evidence that the tRNA population, which is known to be "specialized" for casein synthesis in the mammary gland, is determined as the gland develops and prepares for lactation.

Animals↗
PubMed1985

A human opal suppressor tRNA gene and pseudogene.

A human DNA library, cloned in bacteriophage lambda, was screened with an opal suppressor tRNA probe. Two genes were isolated, subcloned into pBR322, and sequenced. One is a normal opal suppressor tRNA gene 87 nucleotides in length without intervening sequences. It has a TCA anticodon demonstrating that the mature tRNA reads the termination codon UGA. The 5' internal control region for transcription has two extra nucleotides compared to the consensus sequence for eucaryotic tRNA genes, while the 3' internal control region is normal. This gene differs from a previously sequenced chicken opal suppressor serine tRNA gene (Hatfield, D., Dudock, B., and Eden, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4940-4944) only at position 11. The second human gene appears to be a pseudogene truncated near the 3' end, since in the cloverleaf form of the mature tRNA there are three noncomplementary bases in the acceptor stem. The two human genes have a high degree of homology and, excluding the truncated 3' terminus of the pseudogene, differ in only two positions. The flanking sequences of the pseudogene are about 90% homologous to the consensus sequence of the human Alu family of repeated sequences. This gene appears to have been inserted between two adjacent Alu family members.

Animals↗
PubMed1985

Tumor-specific, hypomodified phenylalanyl-tRNA is utilized in translation in preference to the fully modified isoacceptor of normal cells.

Phenylalanine transfer RNA (tRNAPhe) of mammalian tissues contains the hypermodified guanine derivative Y (Wye) adjacent to the 3'-end of the anticodon and two O-methylated bases in the 5' portion of the anticodon loop. These positions are hypomodified in a variety of tumor cells including a mouse neuroblastoma. The normal and tumor-specific Phe-tRNAPhe iso-acceptors were prepared from mouse liver and mouse neuroblastoma cells and compared for their activity in incorporating phenylalanine into each phenylalanine site of rabbit globin in a reticulocyte cell-free protein synthesizing system. The hypomodified Phe-tRNAPhe of neuroblastoma cells is generally preferred to the fully modified tRNAPhe of liver in globin synthesis by about 15%. This preference is the same in the translation of both phenylalanine codons, UUC and UUU, but the ratios of incorporation by the Phe-tRNAPhe species vary from site to site within a 2-fold range. Only 2 of 16 phenylalanine residues are donated preferentially by the fully modified Phe-tRNAPhe. One such residue occurs in beta-42, the second of two tandem phenylalanine residues (both encoded by UUC), while the hypomodified isoacceptor is preferred in translation of the first residue. This result indicates that the translation of tandem residues is particularly affected by the tRNAs available. Since the tumor-specific hypomodified Phe-tRNAPhe is generally utilized preferntially, it appears that the bulky Y base and/or other modifications of normal tRNAPhe may modulate protein synthesis and that tumor cells may achieve a growth advantage if their tRNAPhe is hypomodified.

Animals↗
PubMed1983

Characterization and nucleotide sequence of a chicken gene encoding an opal suppressor tRNA and its flanking DNA segments.

A naturally occurring opal suppressor serine tRNA has been purified from chicken liver and used as a probe to isolate the corresponding gene from a library of chicken DNA in bacteriophage lambda. This minor tRNA is encoded by a single-copy gene that is not part of a tRNA gene cluster. DNA sequence analysis of the gene and its flanking DNA segments shows that the gene is encoded in an 87-base-pair segment without intervening sequences and specifies a tRNA that reads the termination codon UGA. This gene has additional nucleotides in the 5' internal promoter region but has a normal 3' internal promoter sequence and the usual termination signal.

Animals↗