PubMed Health⌕ Search

Biomedical subjects

R T Baker

Publications and source records attributed to R T Baker.

17 recordsLinked to original sources
PubMed1995

Yeast N-terminal amidase. A new enzyme and component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We isolated a Saccharomyces cerevisiae gene, termed NTA1, that encodes an amidase (Nt-amidase) specific for N-terminal asparagine and glutamine. Alterations at the putative active-site cysteine of the 52-kDa Nt-amidase inactivate the enzyme. Null nta1 mutants are viable but unable to degrade N-end rule substrates that bear N-terminal asparagine or glutamine. The effects of overexpressing Nt-amidase and other components of the N-end rule pathway suggest interactions between these components and the existence of a multienzyme targeting complex.

Acyltransferases↗
PubMed1995

Molecular cloning of a cDNA and chromosomal localization of a human theta-class glutathione S-transferase gene (GSTT2) to chromosome 22.

Until recently the Theta-class glutathione S-transferases (GSTs) were largely overlooked due to their low activity with the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) and their failure to bind to immobilized glutathione affinity matrices. Little is known about the number of genes in this class. Recently, Pemble et al. (Biochem J. 300: 271-276, 1994) reported the cDNA cloning of a human Theta-class GST, termed GSTT1. In this study, we describe the molecular cloning of a cDNA encoding a second human Theta-class GST (GSTT2) from a lambda gt11 human liver 5'-stretch cDNA library. The encoded protein contains 244 amino acids and has 78.3% sequence identity with the rat subunit 12 and only 55.0% identity with human GSTT1. GSTT2 has been mapped to chromosome 22 by somatic cell hybrid analysis. The precise position of the gene was localized to subband 22q11.2 by in situ hybridization. The absence of other regions of hybridization suggests that there are no closely related sequences (e.g., reverse transcribed pseudogenes) scattered throughout the genome and that if there are closely related genes, they must be clustered near GSTT2. Southern blot analysis of human DNA digested with BamHI shows that the size of the GSTT2 gene is relatively small, as the coding sequence falls within a 3.6-kb BamHI fragment.

Amino Acid Sequence↗
PubMed1994

Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase.

Expression of cloned genes in prokaryotes such as Escherichia coli is a widely used technique in both basic research and biotechnology. Despite the availability of several E. coli expression vector systems, adequate levels of expression may not be achieved. Expressing proteins as fusions to the highly conserved eukaryotic protein ubiquitin has been reported by several investigators to enhance protein yield in both bacterial and eukaryotic systems. We have modified this technique by the co-expression in E. coli of a ubiquitin-fusion protein and the Saccharomyces cerevisiae ubiquitin-specific protease Ubp2. This allows the co-translational cleavage of engineered ubiquitin-fusion proteins expressed in E. coli. This system was used to express a human Pi class glutathione S-transferase (GST) GSTP1 as well as two mutant GSTP1 derivatives, Trp39-->Cys and Gln52-->Glu. The yield of these enzymes was improved 40-fold by using the ubiquitin-fusion/co-translational cleavage technique, and no uncleaved product was detected. The Trp39-->Cys mutant was totally devoid of GST activity, while the activity of the Gln52-->Glu mutant was reduced to 6% of wild-type GSTP1-1. As both of the mutated residues map within the glutathione-binding site, the reduced GST activity is consistent with a marked reduction in glutathione binding ability.

Amino Acid Sequence↗
PubMed1994

Localization of the human UBA52 ubiquitin fusion gene to chromosome band 19p13.1-p12.

Because of the conservation of the ubiquitin coding sequence and the number of transcriptionally active genes and reverse-transcribed pseudogenes, it has not been possible to use ubiquitin cDNA clones to map the functional ubiquitin genes. The UBB and UBC polyubiquitin genes have previously been mapped by the use of specific intron or 5' flanking sequence probes. In this study, we have used an intron sequence from the UBA52 gene for chromosome mapping studies. Analysis of somatic cell hybrids containing individual human chromosomes indicated that the UBA52 gene is located on chromosome 19. In situ hybridization studies confirmed the chromosomal localization but showed two peaks of hybridization: a major one over 19p13.1-p12 and a secondary one over 19q12-q13.11. Because the peak of hybridization over 19p13.1-p12 was consistently the strongest in five individuals, it is likely that this is the location of the UBA52 gene. Thus far, three of the four transcriptionally active ubiquitin genes have been assigned to separate chromosomes.

Animals↗
PubMed1992

Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family.

In eukaryotes, both natural and engineered ubiquitin (Ub) fusions to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. YUH1 and UBP1, the genes for two ubiquitin-specific proteases of the yeast Saccharomyces cerevisiae, have been cloned previously and shown to encode nonhomologous proteins. Using an Escherichia coli-based genetic screen, we have isolated two other yeast genes for ubiquitin-specific proteases, named UBP2 and UBP3. Ubp2 (1,264 residues), Ubp3 (912 residues), and the previously cloned Ubp1 (809 residues) are largely dissimilar except for two short regions containing Cys and His which encompass their putative active sites. Neither of these proteases has sequence similarities to Yuh1. Both Ubp2 and the previously identified Ubp1 cleave in vitro at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size, poly-Ub being the exception. However, both Ubp1 and Ubp2 are also capable of cleaving poly-Ub when coexpressed with it in E. coli, suggesting that such cleavage is largely cotranslational. Although inactive in E. coli extracts, Ubp3 was active with all of the tested ubiquitin fusions except poly-Ub when coexpressed with them in E. coli. Null yuh1 ubp1 ubp2 ubp3 quadruple mutants are viable and retain the ability to deubiquitinate ubiquitin fusions, indicating the presence of at least one more ubiquitin-specific processing protease in S. cerevisiae.

Amino Acid Sequence↗
PubMed1992

Localization of the human UBC polyubiquitin gene to chromosome band 12q24.3.

The localization of specific human ubiquitin genes has not been straightforward because of the conservation of the ubiquitin coding sequence and the number of processed pseudogenes. An congruent to 1.4-kb sequence from the 5'-flanking region of the UBC gene has been shown to be unique to that locus and free from dispersed repeat elements. The cloned 5'-flanking fragment has been used to probe Southern blots of DNA obtained from somatic cell hybrid cell lines. These data indicate that the UBC gene is located on chromosome 12. In situ hybridization with the 5'-flanking probe has refined the assignment to the broad chromosomal subband 12q24.3. These data show that the active ubiquitin genes are not clustered and are located on separate chromosomes. In addition, these studies demonstrate the utility of intron or flanking sequence probes in the specific chromosomal assignment of members of highly conserved gene families.

Chromosome Mapping↗
PubMed1992

The human ubiquitin/52-residue ribosomal protein fusion gene subfamily (UbA52) is composed primarily of processed pseudogenes.

Two members of the human ubiquitin gene subfamily encoding ubiquitin fused to a 52-residue ribosomal protein (UbA52) were cloned, sequenced, and analyzed. Both were found to be processed (reverse-transcribed) pseudogenes, containing several nucleotide substitutions and deletions that severely disrupt their potential open-reading frames. Genomic hybridization analysis using probes derived from a functional UbA52 gene (UBA52) suggests that the UbA52 subfamily consists of one intron-containing, transcriptionally active gene and approximately eight processed pseudogenes, a feature characteristic of mammalian ribosomal protein gene families.

Base Sequence↗
PubMed1991

The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.

Complementary DNA clones encoding ubiquitin fused to a 52 amino acid tail protein were isolated from human placental and adrenal gland cDNA libraries. The deduced human 52 amino acid tail protein is very similar to the homologous protein from other species, including the conservation of the putative metal-binding, nucleic acid-binding domain observed in these proteins. Northern blot analysis with a tail-specific probe indicated that the previously identified UbA mRNA species most likely represents comigrating transcripts of the 52 amino acid tail (UbA52) and 80 amino acid tail (UbA80) ubiquitin fusion genes. The UbA52 gene was isolated from a human genomic library and consists of five exons distributed over 3400 base pairs. One intron is in the 5' non-coding region, two interrupt the single ubiquitin coding unit, and the fourth intron is within the tail coding region. Several members of the Alu family of repetitive DNA are associated with the gene. The UbA52 promoter has several features in common with mammalian ribosomal protein genes, including its location in a CpG-rich island, initiation of transcription within a polypyrimidine tract, the lack of a consensus TATA motif, and the presence of Sp1 binding sites, observations that are consistent with the recent identification of the ubiquitin-free tail proteins as ribosomal proteins. Thus, in spite of its unusual feature of being translationally fused to ubiquitin, the 52 amino acid tail ribosomal protein is expressed from a structurally typical ribosomal protein gene.

Adrenal Glands↗
PubMed1991

Inhibition of the N-end rule pathway in living cells.

The N-end rule relates the metabolic stability of a protein to the identify of its amino-terminal residue. Previous work, using amino acid derivatives such as dipeptides to inhibit N-end rule-mediated protein degradation in an extract from mammalian reticulocytes, has demonstrated the existence of specific N-end-recognizing proteins in this in vitro system. We now show that these nontoxic amino acid derivatives, when added to growing cells of the yeast Saccharomyces cerevisiae, are able to inhibit the degradation of proteins by the N-end rule pathway in vivo. Moreover, this inhibition is shown to be selective for the two distinct classes of destabilizing amino-terminal residues in substrates of the N-end rule pathway.

Amino Acids↗
PubMed1990

Localization of the human UbB polyubiquitin gene to chromosome band 17p11.1-17p12.

The chromosomal location of the human ubiquitin genes has been evaluated by in situ hybridization. Because of the conservation of the ubiquitin sequence, coding-region probes cannot distinguish between specific ubiquitin genes and reveal ubiquitin sequences in a number of different chromosomal regions. The major sites of hybridization with a coding-region probe include 17p11.1-p12, 12p24.2-q24.32, and 2q21-q24, with weaker hybridization over 1p3, 1q4, 2q3, and 13q. Hybridization with a probe isolated from the UbB gene intron indicated that this gene is located within the region 17p11.1-17p12. This region showed the strongest hybridization with the coding-region probe and is presumably also the location of the duplicated UbB pseudogene.

Blotting, Southern↗
PubMed1990

The adrenal response to trauma, operation and cosyntropin stimulation.

Thirty-four patients who underwent abdominal or thoracic, or both, operation for injuries were studied. Serum and urinary cortisol levels were elevated perioperatively and returned to normal levels by the fourth postoperative day. Postoperative complication was associated with persistent elevation of levels of serum and urinary cortisol. Baseline serum cortisol levels correlated with Injury Severity Scores. Cosyntropin stimulation produced a significant rise in serum cortisol to a peak level of 47.4 +/- 10.2 (S.D.) micrograms per deciliter in patients after emergent operations for traumatic injury. Use of the cosyntropin stimulation test for the diagnosis of postoperative adrenal insufficiency can be extended to patients who undergo operation for traumatic injury.

Adrenal Glands↗
PubMed1989

Unequal crossover generates variation in ubiquitin coding unit number at the human UbC polyubiquitin locus.

An observed mRNA length polymorphism of the human UbC polyubiquitin gene transcript was shown to correlate exactly with a three-allele HaeIII RFLP. Both polymorphisms apparently result from a variation in the number of ubiquitin coding units per UbC allele. Transcriptionally active alleles containing seven, eight, or nine coding units were observed, at frequencies suggesting that alleles of higher coding-unit number are selectively retained in the population. We propose unequal crossover events, promoted by the highly repetitive structure of the polyubiquitin gene, as a basis for the coding-unit number variation, and we present preliminary evidence for such a crossover, on the basis of analysis of known UbC DNA sequences.

Biological Evolution↗
PubMed1987

The human ubiquitin gene family: structure of a gene and pseudogenes from the Ub B subfamily.

An ubiquitin cDNA clone was isolated from a human liver cDNA library. This clone contained two complete, and a portion of a third, ubiquitin coding sequences joined head to tail with no spacer peptides. Screening a human genomic library with a probe derived from the coding region of this cDNA identified a large number of cross-hybridising clones. Differential screening of these genomic clones with the 3' non-coding region of the cDNA identified three different 3'-positive clones. Sequence analysis of these three clones revealed: a gene corresponding to the cDNA containing an intron in the 5' non-coding region and coding for three direct repeats of mature ubiquitin, and two related pseudogenes which appear to have arisen by reverse transcription and insertion into the genome. However, one pseudogene contains two repeats of the ubiquitin coding sequence, while the other contains only one. Hybridisation analysis of restricted human genomic DNA suggests the presence of one other closely related gene within the genome.

Amino Acid Sequence↗
PubMed1981

Operant escape learning in decerebrate duck embryos.

Peking duck embryos were trained in an operant escape task on Day 25 of incubation (total incubation period = 27 days) following decerebration on Day 24 of incubation. Experimental embryos successfully acquired the operant response, which involved the performance of a discrete foot flexion in order to terminate a shock applied to the wing. The decerebrate embryos resembled normal, unoperated embryos of this age in their acquisition of the operant behavior. This study demonstrates that this form of learning can be acquired without benefit of telencephalic structures, at least at this point in development. Differences in the consequences of decerebration during prenatal or neonatal versus adult stages are discussed.

Animals↗