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Biomedical subjects

D Hatfield

Publications and source records attributed to D Hatfield.

At least 19 recordsLinked to original sources
PubMed2000

Yeast asparagine (Asn) tRNA without Q base promotes eukaryotic frameshifting more efficiently than mammalian Asn tRNAs with or without Q base.

In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting. Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNA(ASn)(-Q) promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts. The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule. However, we cannot absolutely rule out a role of Q base in frameshifting as wheat germ extracts and a lysate depleted of most of its tRNA and supplemented with calf liver tRNA contain both Asn tRNA with or without Q base.

Animals↗
PubMed1997

Selenium supplementation suppresses tumor necrosis factor alpha-induced human immunodeficiency virus type 1 replication in vitro.

Selenium is a nutritionally essential trace element that is important for optimal function of the immune system. It is incorporated into selenoproteins as the amino acid selenocysteine and it is known to inhibit the expression of some viruses. In this study, we show that selenium supplementation for 3 days prior to exposure to tumor necrosis factor alpha (TNF-alpha) partially suppresses the induction of human immunodeficiency virus type 1 (HIV-1) replication in both chronically infected T lymphocytic and monocytic cell lines. In acute HIV-1 infection of T lymphocytes and monocytes in the absence of exogenous TNF-alpha, the suppressive effect of selenium supplementation was not observed. However, selenium supplementation did suppress the enhancing effect of TNF-alpha on HIV-1 replication in vitro in acutely infected human monocytes, but not in T lymphocytes. Selenium supplementation also increased the activities of the selenoproteins, glutathione peroxidase (GPx) and thioredoxin reductase (TR), which serve as cellular antioxidants. Taken together, these results suggest that selenium supplementation may prove beneficial as an adjuvant therapy for AIDS through reinforcement of endogenous antioxidative systems.

Cells, Cultured↗
PubMed1995

Identification of new selenocysteine tRNA[SER]SEC isoacceptors in human cell lines.

The selenocysteine tRNA population was examined in a human T-cell line and in a human monocytic cell line for the occurrence of additional species of selenocysteine tRNA. At least three additional (and possibly more) selenocysteine isoacceptors were found which occur in minor levels as compared to the two major selenocysteine isoacceptors previously characterized. The possible significance of these newly observed species are discussed.

Cell Line↗
PubMed1992

A pseudogene for human glutathione peroxidase.

Glutathione peroxidases (GPx) serve a bioprotective function in the reduction of peroxides to less toxic substances. Both cellular and secreted forms of the protein have been reported, as well a number of distinct cDNA sequences. Previous efforts have described three distinct loci on human chromosomes 3, 21 and X which hybridize to a GPX cDNA and these authors have speculated that only the chromosome 3 locus encodes a functional GPX gene. This conclusion was based on mapping studies showing a precise deletion of intron sequences in the GPX loci on chromosomes 21 and X despite strong conservation among these sequences in both the coding and 3'-untranslated regions. To pursue this issue, we have isolated the chromosome 21 GPX locus by molecular cloning and determined its nucleotide sequence. Consistent with the expectations of McBride et al. [Biofactors 4 (1988) 285-292], the sequence does reveal a highly conserved processed pseudogene. It is suggested that a retrotransposed copy of the GPX gene integrated into chromosome 21 and may have maintained activity prior to the accumulation of inactivating mutations.

Base Sequence↗
PubMed1992

Selenocysteyl-tRNAs recognize UGA in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus.

Selenocysteyl-tRNAs that decode UGA were previously identified in representatives of three of the five life kingdoms which were the monera, animal and protist kingdoms. In the present study, we show that these tRNAs also occur in representatives of the two remaining kingdoms, plants and fungi; i.e., selenocysteyl-tRNAs which code for UGA occur in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus. The fact that selenocysteyl-tRNAs are present in all five life kingdoms strongly suggests that UGA, in addition to dictating the cessation of protein synthesis, also codes for selenocysteine in the universal genetic code.

Base Sequence↗
PubMed1992

Cardiac cycle length variability in ponies at rest and during exercise.

We evaluated cardiac cycle length variability in ponies at rest and during strenuous exercise with and without premedication with atropine. In the absence of premedication, cardiac cycle length at rest was 1,112 +/- 53 ms, the individual cardiac cycle length standard deviation (SDCL) was 75 +/- 23 ms, and the individual cycle length coefficient of variation (CVCL) was 6.32 +/- 1.62. Exercise significantly decreased (P < 0.05) all three indexes (290 +/- 9 ms, 5 +/- 1 ms, and 1.65 +/- 0.20, respectively). Atropine premedication significantly reduced resting cardiac cycle length (685 +/- 46 ms), SDCL (10 +/- 2 ms), and CVCL (1.45 +/- 0.19) compared with nonpremedicated values. Cardiac cycle length was significantly decreased by exercise after atropine premedication, but no statistically significant changes occurred in SDCL or CVCL. Thus, although considerable cardiac cycle length variability exists in nonpremedicated ponies at rest, it is nearly completely abolished by strenuous exercise. The absence of significant differences between the indexes of variability during exercise without premedication, at rest after atropine, and during exercise after atropine indicates that cardiac cycle length variability in the pony is mediated primarily through activity of the parasympathetic system.

Animals↗
PubMed1992

Regional localization of the selenocysteine tRNA gene (TRSP) on human chromosome 19.

The human selenocysteine tRNA gene (TRSP) has been localized on chromosome 19q13.2-->q13.3 by in situ hybridization and ordered with respect to other genes and anonymous DNA markers in this region by linkage analysis in the forty CEPH pedigrees. These loci span only 10 cM in males and about 30 cM in females. The order of the loci is cen ... D19S7-D19S9-D19S47-CYP2A-CYP2F1-APOC2++ +-(TRSP, CKM). CYP2B flanks the CYP2A and CYP2F1 loci, but it cannot be determined whether it is proximal or distal to the other two cytochrome P450 loci with respect to the centromere.

Chromosome Banding↗
PubMed1991

Selenium induces changes in the selenocysteine tRNA[Ser]Sec population in mammalian cells.

Two isoacceptors of selenocysteine tRNA[Ser]Sec are present in higher vertebrates which are responsible for donating selenocysteine to protein. One such selenocysteine containing protein, glutathione peroxidase, requires selenium for its translation and transcription. Since tRNA[Ser]Sec is a critical component of the glutathione peroxidase translational machinery, the levels and distributions of its isoacceptors were examined from both human and rat cells grown in chemically defined media with and without selenium. Not only did the level of the selenocysteine tRNA[Ser]Sec population increase approximately 20% in cells grown in the presence of selenium, but the distributions of the two isoacceptors also changed relative to each other.

Animals↗
PubMed1990

The where, what and how of ribosomal frameshifting in retroviral protein synthesis.

The gag and pol genes of most retroviruses occur in different reading frames and their translation as a single polypeptide is carried out by ribosomal frameshifting in the -1 direction. The alignment of the different reading frames occurs by overlapping reading in response to at least two signals within the RNA: one is a heptanucleotide stretch at the frameshift site and the other is a stem-loop structure which occurs just downstream of the first signal.

Protein Biosynthesis↗
PubMed1990

Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom.

Recently, a mammalian tRNA which was previously designated as an opal suppressor seryl-tRNA and phosphoseryl-tRNA was shown to be a selenocysteyl-tRNA (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield, J. Biol. Chem. 264:9724-9727, 1989). Hence, this tRNA is now designated as selenocysteyl-tRNA[Ser]Sec, and its function is twofold, to serve as (i) a carrier molecule upon which selenocysteine is biosynthesized and (ii) as a donor of selenocysteine, which is the 21st naturally occurring amino acid of protein, to the nascent polypeptide chain in response to specific UGA codons. In the present study, the selenocysteine tRNA gene was sequenced from Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. The tRNA product of this gene was also identified within the seryl-tRNA population of a number of higher and lower animals, and the human tRNA[Ser]Sec gene was used as a probe to identify homologous sequences within genomic DNAs of organisms throughout the animal kingdom. The studies showed that the tRNA[Ser]Sec gene has undergone evolutionary change and that it is ubiquitous in the animal kingdom. Further, we conclude that selenocysteine-containing proteins, as well as the use of UGA as a codon for selenocysteine, are far more widespread in nature than previously thought.

Animals↗
PubMed1989

Transcription of Xenopus selenocysteine tRNA Ser (formerly designated opal suppressor phosphoserine tRNA) gene is directed by multiple 5'-extragenic regulatory elements.

A tRNA gene whose product is aminoacylated with serine and the serine moiety is then phosphorylated to form phosphoseryl-tRNA (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review) has now been shown to form selenocysteyl-tRNA; hence the corresponding gene is designated as selenocysteine tRNA Ser (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield (1989) J. Biol. Chem. 264, in press). In the present study, we show that the expression of this unique tRNA gene is governed by at least three upstream regulatory elements. In initial studies, the relative efficiencies of transcription of the human, rabbit, chicken, and Xenopus selenocysteine tRNA genes were compared in vivo in Xenopus oocytes and in vitro in HeLa cell extracts. The Xenopus gene was severalfold more actively expressed, both in vivo and in vitro, than the human and rabbit genes, whereas the chicken gene was poorly expressed. Exchange of the 5'-flanking regions of the Xenopus and chicken genes, which have identical gene sequences, reversed their levels of transcription, demonstrating that a regulatory site or sites exist upstream of these genes. Deletion-substitution mutants in the Xenopus gene and its 5'-flanking sequence show in in vitro assays that 1) the level of transcription is reduced substantially when a GC-rich stretch that is immediately upstream of a TATA box in the -30 region is removed; 2) the level of transcription is virtually abolished when the TATA box is removed; and 3) deletions up to and further upstream of the GC-rich region do not affect the level of transcription. The same deletions, when used in in vivo assays, demonstrate a step-down in expression with the deletion removing the GC-rich region, a further step-down in expression with the deletion removing the TATA box, but the most pronounced reduction in expression was observed with a deletion removing an AT-rich region between nucleotides -62 and -76. Thus, a regulatory site was identified in vivo which was not detected in vitro, and transcription of the selenocysteine tRNA Ser gene is determined by multiple upstream regulatory elements.

Amino Acid Sequence↗
PubMed1989

Chromatographic analysis of the aminoacyl-tRNAs which are required for translation of codons at and around the ribosomal frameshift sites of HIV, HTLV-1, and BLV.

An examination of the frameshift signals or proposed signals within published sequences of retroviruses and other genetic elements from higher animals shows that each site utilizes a tRNA which normally contains Wybutoxine (Wye) base or Queuine (Q) base in the anticodon loop. We find experimentally that most of the Phe-tRNA present in HIV-1 infected cells lacks the highly modified Wye base in its anticodon loop and most of the Asn-tRNA in HTLV-1 and BLV infected cells lacks the highly modified Q base in its anticodon loop. Interestingly, Phe-tRNA translates a UUU codon within the ribosomal frameshift signal in HIV and Asn-tRNA translates a AAC codon within the proposed frameshift signals in HTLV-1 and BLV. Thus, the lack of a highly modified base in the anticodon loop of tRNAs in retroviral infected cells is correlated with the participation of these undermodified tRNAs in the corresponding frameshift event. This suggests that the "shifty" tRNAs proposed by Jacks et al. (Cell 55, 447-458, 1988) to carry out frameshifting may be hypomodified isoacceptors.

Animals↗
PubMed1988

Immunopurification of the suppressor tRNA dependent rabbit beta-globin readthrough protein.

In mammalian cells, the rabbit beta-globin readthrough protein is the only known example of a naturally occurring readthrough protein which does not involve a viral system. To provide an efficient means for its isolation, detection, and study, we elicited specific antibodies against this unique protein. The 22 amino acid peptide corresponding to the readthrough portion of this protein was synthesized, coupled to keyhole limpet hemocyanin, and injected into sheep. Specific antibodies to the peptide were produced as demonstrated by the enzyme-linked immunosorbent assay technique and by immunoblotting. The antibodies did not react with globin. The rabbit beta-globin readthrough protein was separated from globin and other reticulocyte proteins by polyacrylamide gel electrophoresis and visualized by silver staining or by labeling with [35S]methionine. Incorporation of [35S]methionine into the readthrough protein was significantly enhanced upon addition of an opal suppressor tRNA to reticulocyte lysates. Immunoblotting revealed that the readthrough protein also occurs in lysates without added suppressor tRNA. The antibodies were purified on an affi-gel column which had been coupled with the peptide antigen. The readthrough protein was then purified from reticulocytes by immunoaffinity chromatography and by high-performance liquid chromatography. The results provide conclusive evidence that the beta-globin readthrough protein is naturally occurring in rabbit reticulocytes.

Amino Acid Sequence↗
PubMed1988

Effect of feeding on renin-angiotensin-aldosterone system of the horse.

The effect of feeding frequency and associated meal size on the renin-angiotensin-aldosterone system (RAAS) in seven horses was examined. A daily maintenance ration of hay-grain pellets was provided either as a multiple feeding regimen (MF), in which the ration was divided into six equal portions fed at 4-h intervals, or as a single large feeding (SF) given from 9 A.M. until 11 A.M. Plasma renin activity (PRA), aldosterone (PAC), cortisol (PCC), protein concentration (TP), packed cell volume (PCV), and serum sodium and potassium were measured serially. To prevent significant RAAS stimulation due to strenuous exercise or by assuming orthostatism after a period of recumbency, the horses were trained to stand in 1 X 4-m tie stalls during the experiments. Changes in Na intake were prevented by disallowing nonration salt sources. A 12:12 light-dark interval was maintained. During the MF experiment, only serum Na changed diurnally, with concentrations lowest in early morning and highest before midday. In contrast, during the SF experiment, PRA was increased at 0.5, 1.0, and 3.0 h and PAC was increased at 3.0, 5.0, and 7.0 h after onset of feeding (P less than 0.005). Increased TP and PCV suggested transient hypovolemia was responsible for renin release. Significant increases in Na and decreases in K occurred while eating; however, K increased postprandially to be coincident with aldosterone. Except for a transient increase during feeding in SF, PCC demonstrated a similar circadian rhythm in both experiments. It was concluded that 1) episodic feeding (SF) causes significant diurnal variation of the RAAS in the horse, and 2) spontaneous circadian activity of the RAAS cannot be demonstrated in this species during a steady-state feeding regimen (MF).

Aldosterone↗
PubMed1988

Gene for selenium-dependent glutathione peroxidase maps to human chromosomes 3, 21 and X.

A human glutathione peroxidase cDNA has been used as a probe to hybridize to DNAs isolated from human - rodent somatic cell hybrids that have segregated human chromosomes. A 609 bp probe which contains the entire coding region hybridizes to human chromosomes 3, 21 and Xp. Fragments of the cDNA coding sequence and of the 3' untranslated region were also used as probes. These fragments hybridized to each of the three chromosomes with the same efficiency, suggesting similarity between the loci, whereas an intronic probe detected only the gene on chromosome 3. The general organization of each gene was determined from the hybridization data. The data suggest that the locus on chromosome 3 is a functional gene containing a single intron and a pattern of restriction sites identical to those found in the cDNA coding sequence. The data also suggest that the sequences on chromosomes X and 21 have equal conservation of the 3' untranslated and coding sequences but do not contain introns, providing evidence that the latter two sequences are processed pseudogenes. A simple two allele polymorphism in PvuII digests was detected at the locus on chromosome 21.

Animals↗
PubMed1987

Opal suppressor phosphoserine tRNA gene and pseudogene are located on human chromosomes 19 and 22, respectively.

An opal suppressor phosphoserine tRNA gene and pseudogene have been isolated from a human DNA library and sequenced (O'Neill, V., Eden, F., Pratt, K., and Hatfield, D. (1985) J. Biol. Chem. 260, 2501-2508). Southern hybridization of human genomic DNA with an opal suppressor tRNA probe suggested that the gene and pseudogene are present in single copy. In this study, we have determined the chromosome location of the human gene and pseudogene by utilizing a 193-base pair fragment encoding the opal suppressor phosphoserine tRNA gene as probe to examine DNAs isolated from human-rodent somatic cell hybrids that have segregated human chromosomes. These studies show that the probe hybridized with two regions in the human genome; one is located on chromosome 19 and the second on chromosome 22. By comparing the restriction sites within these two regions to those previously determined for the human opal suppressor phosphoserine tRNA gene and pseudogene, we tentatively assigned the gene to chromosome 19 and the pseudogene to chromosome 22. These assignments were confirmed by utilizing a 350-base pair fragment which was isolated from the 5'-flanking region of the human gene as probe. This fragment hybridized only to chromosome 19, demonstrating unequivocally that the opal suppressor phosphoserine tRNA gene is located on chromosome 19. The flanking probe hybridized to a single homologous band in hamster and in mouse DNA to which the gene probe also hybridized, demonstrating that the 5'-flanking region of the opal suppressor tRNA gene is conserved in mammals. Restriction analysis of DNAs obtained from the white blood cells of 10 separate individuals demonstrates that the gene is polymorphic. This study provides two additional markers for the human genome and constitutes only the second set of two tRNA genes assigned to human chromosomes.

Animals↗