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Biomedical subjects

G J Goodall

Publications and source records attributed to G J Goodall.

36 records · Page 2Linked to original sources
PubMed1992

Three essential promoter elements mediate tumour necrosis factor and interleukin-1 activation of the granulocyte-colony stimulating factor gene.

Granulocyte-colony stimulating factor (G-CSF) is a haemopoietic growth factor produced by mesenchymal cells but not T lymphocytes after stimulation with specific cytokines or mitogens. A 330 bp promoter fragment of the human G-CSF gene induced reporter gene expression in human embryonic lung fibroblasts in response to tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). The same promoter fragment was not active in Jurkat T cells nor did it respond to phorbol ester in either cell type. At least three distinct elements, the CK-1 sequence, a decanucleotide present in haemopoietic growth factor genes, an NF-IL-6 consensus sequence and a consensus octamer sequence, were essential in the G-CSF promoter for TNF-alpha and IL-1 beta response. Mutation of any of these sequences abolished promoter function. In contrast, mutation of two other consensus protein binding sequences, i.e. a Pu-1 site and a CK-2-like sequence, did not eliminate promoter function. Both the CK-1 and octamer sequences acted independently as TNF-alpha and IL-1 beta responsive elements upstream of a heterologous promoter. The response of the octamer sequence and the 330 bp promoter but not the CK-1 sequence was greater with IL-1 beta than TNF-alpha reflecting a similar response of the endogenous gene.

Base Sequence↗
PubMed1991

Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.

We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.

Base Sequence↗
PubMed1990

The minimum functional length of pre-mRNA introns in monocots and dicots.

When exogenous genes are to be expressed in transgenic plants, their RNAs must be correctly processed. To gain information useful for predicting whether foreign introns will be accurately spliced, we have analysed the processing of an artificial gene in maize and Nicotiana plumbaginifolia protoplasts. A synthetic plant intron, devised to contain the elements necessary for pre-mRNA splicing in dicots, was found to be efficiently spliced in a monocot (maize) transient expression system. A series of deletion mutants of the synthetic intron was constructed to assess the minimum functional intron length. In both monocots and dicots this was found to be between 70 and 73 nt. This length requirement is similar to that seen in vertebrates, but significantly greater than that in fungi and insects.

Base Sequence↗
PubMed1990

Recognition efficiency of Dicotyledoneae-specific promoter and RNA processing signals in rice.

Heterologous gene expression experiments have shown that genes of Monocotyledoneae are often not transcribed in Dicotyledoneae, or produce pre-mRNA that is inefficiently or aberrantly processed. It is however not known how correctly and efficiently dicotyledon-specific gene expression signals are recognized in cells of Monocotyledoneae. Here we address this question using tobacco (Nicotiana tabacum) and rice (Oryza sativa) protoplasts transformed with the same hybrid gene constructs. Constructs including the nptII protein coding sequence fused to Cauliflower Mosaic Virus (CaMV) promoter and polyadenylation signals were used to obtain stably transformed cell lines of tobacco and rice. In one of the constructs the nptII coding region is interrupted by a modified intron-3 sequence from the soybean phaseolin gene. Although the mean number of hybrid gene copies integrated into the rice genome was on average 5- to 10-fold higher than in tobacco, the steady-state transcript level was 3 times lower. A lower level of transcript was also observed in transient expression experiments. The amount of the mature mRNA was not influenced by the presence of the intron. The phaseolin intron was processed in rice with high efficiency and an accuracy indistinguishable from that seen in tobacco.

Base Sequence↗
PubMed1989

The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing.

Plant cells do not in general process the introns of transcripts expressed from introduced vertebrate genes. By studying the processing of model introns in transfected plant protoplasts, we have investigated the special requirements for intron recognition by plant cells. Our results indicate that the requirements for intron recognition in plants are different from those of both metazoa and yeast. A synthetic intron of arbitrary sequence but incorporating splice site consensus sequences and a high proportion of U and A nucleotides, a characteristic feature of plant introns, was efficiently spliced in protoplasts. We have studied the effects of various sequence alterations and conclude that AU-rich sequences are necessary for intron recognition. In addition, we find that the criteria for branch site selection are relaxed, as they are in vertebrates, but a polypyrimidine tract is not necessary.

Base Sequence↗
PubMed1988

Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs.

A rat spleen cDNA library was screened for clones carrying the cDNAs for prothymosin alpha and parathymosin. Sequence analysis of a clone carrying the entire coding region for prothymosin alpha confirmed and completed the amino acid sequence for this polypeptide and established the number of amino acid residues as 111. Rat prothymosin alpha differs from human prothymosin alpha at six positions, including four substitutions and two insertions. The nucleotide sequences of the cDNAs for the rat and human polypeptides are more than 90% identical in the open reading frames, with significant homology extending into the 5' and 3' flanking regions. From the same library, we also isolated a clone carrying 80% of the coding region for rat parathymosin. The number of amino acid residues in rat parathymosin is 101, based on the sequence deduced from the cDNA insert and earlier information on the sequence in the amino-terminal portion of this polypeptide. Despite their similarity in size and amino acid composition, rat prothymosin alpha and rat parathymosin show only limited sequence homology, primarily in the segment including residues 14 through 25, where 10 of 12 positions are identical in the two polypeptides. this is also the region of significant sequence similarity to a 12-amino-acid segment in the p17 protein of the human immunodeficiency disease associated virus (HTLV-IIIB).

Amino Acid Sequence↗
PubMed1987

Molecular cloning of the cDNA for rat spleen thymosin beta 10 and the deduced amino acid sequence.

A rat spleen cDNA library was prepared and employed for the molecular cloning of the cDNA for thymosin beta 10, a peptide that previously had been found to accompany the closely related peptide, thymosin beta 4, in several species of mammals (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B. L. Horecker (1983) Arch. Biochem. Biophys. 225, 407-413). First-round screening with a synthetic oligodeoxynucleotide probe yielded 55 positive clones, and sequence analysis of 11 of these clones revealed that they all coded for a peptide containing the thymosin beta 10 sequence, except for an additional arginyl residue at position 39. This peptide, designated thymosin beta 10arg, had been identified previously in rabbit tissues and reported as a variant of thymosin beta 10 (S. Ruggieri, S. Erickson-Viitanen, and B.L. Horecker (1983) Arch. Biochem. Biophys. 226, 388-392). Analysis of the 55 positive clones using a specific oligodeoxynucleotide probe constructed to correspond to the mRNA sequence, including the codon for Arg39, confirmed that they all coded for the amino acid sequence including Arg39. Based on these results, the existence of a molecular species lacking Arg39 is considered unlikely, and we conclude that thymosin beta 10 contains 43, rather than 42, amino acid residues, with identity to thymosin beta 4 in 32 of the 43 residues. We propose that the name thymosin beta 10 be used to refer to the peptide containing Arg39 and that the designation thymosin beta 10arg be dropped. In the cDNA sequence the codons for Ala1 and Ser43 of thymosin beta 10 are flanked by initiator and terminator codons, respectively; thus, both the thymosin beta 4 and thymosin beta 10, which coexist in mammalian cells and tissues, are synthesized without the formation of larger polypeptide precursors.

Amino Acid Sequence↗
PubMed1986

Molecular cloning of cDNA for human prothymosin alpha.

A cDNA library was constructed from human spleen mRNA and screened for clones containing cDNAs coding for prothymosin alpha. A clone containing a 503-base-pair insert including the entire coding sequence for the translated portion of the mRNA was isolated. The deduced amino acid sequence confirms and completes the partial sequence of human prothymosin alpha determined by protein sequencing methods. The presence of an initiator codon immediately preceding the codon for the NH2-terminal serine residue and of a terminator codon immediately following the codon for Asp-109, the COOH-terminal residue, suggests that prothymosin alpha is synthesized without formation of a larger precursor polypeptide. Analysis of the 5' sequence preceding the initiator methionine codon excluded the presence of a signal peptide in the translated sequence.

Amino Acid Sequence↗
PubMed1985

Sequence of a cloned 523-bp cDNA for thymosin beta 4.

The sequence of a 523-bp cDNA, isolated from a clone bank prepared from partially purified rat spleen mRNA coding for thymosin beta 4, was described. The 3' sequence extended through the poly(A) segment and the 5' sequence included 36 bp preceding the translated sequence. The putative amino acid sequence coded by this segment possesses some of the features of a signal peptide.

Amino Acid Sequence↗
PubMed1985

Pyruvate carboxylase: mechanisms of the partial reactions.

Data from isotopic exchange studies and from experiments with 32P- and 14C-labeled enzyme-bound intermediates support the following description of the first partial reaction: (Formula: see text). From studies of the transfer of the carboxyl-group from ENZ-biotin-CO2- to pyruvate or its analogues we propose that binding of the acceptor substrate induces the translation of carboxybiotin from the first to the second partial reaction site. The studies on the translocation of carboxybiotin can be summarized in the following reaction scheme: (Formula: see text). Where k+3 less than k+1, k-1, k+2 and k-2. Thus, the rate-limited step is governed by k+3 which represents the movement of carboxybiotin from the first subsite to the second.

Acetyl Coenzyme A↗
PubMed1984

Prothymosin alpha: isolation and properties of the major immunoreactive form of thymosin alpha 1 in rat thymus.

A polypeptide containing approximately equal to 112 amino acid residues, with the thymosin alpha 1 sequence at its NH2 terminus, has been isolated from rat thymus by using a radioimmunoassay with an antibody prepared against synthetic thymosin alpha 1. The new polypeptide, named "prothymosin alpha," was found to be the major substance crossreacting with thymosin alpha 1 antiserum in rat thymus extracts; peptides corresponding to thymosin alpha 1 or thymosin alpha 11 were not detected. In gel filtration at pH 2.8, prothymosin alpha emerged as a single symmetrical peak corresponding to an apparent molecular weight of 32,000, approximately 3 times larger than the minimum molecular weight calculated from its amino acid composition. On the same gel filtration columns, synthetic thymosin alpha 1 (calculated Mr = 3108) emerged at a position corresponding to a molecular weight of 10,000-11,000. Thus, both prothymosin alpha and thymosin alpha 1 appear to exist in solution as oligomers, possibly as trimers. Prothymosin alpha and synthetic thymosin alpha 1 also were separated readily in reverse-phase HPLC and in isoelectric focusing; the isoelectric point of prothymosin alpha determined by the latter procedure was found to be 3.55, consistent with an unusually high content of glutamic and aspartic acids based on amino acid analyses. Prothymosin alpha appears to represent the native polypeptide from which thymosin alpha 1 and other fragments are generated during the isolation of thymosin fraction 5.

Amino Acid Sequence↗
PubMed1983

A mechanism for the transfer of the carboxyl-group from 1'-N-carboxybiotin to acceptor substrates by biotin-containing enzymes.

Previous proposals for the mechanism by which biotin-dependent enzymes catalyse the transfer of the carboxyl group from 1'-N-carboxybiotin to acceptor molecules do not appear to be consistent with all of the experimental observations now available. We propose a multi-step mechanism in which (a) substrate and then carboxybiotin bind at the second partial reaction site, (b) a base positioned adjacent to the 3'-N of the carboxybiotin abstracts a proton from the 3'-N and (c) the resulting enolate ion and the acceptor substrate undergo a concerted reaction resulting in carboxyl-group transfer.

Binding Sites↗
PubMed1983

Thymosin beta 4 in cultured mammalian cell lines.

Thymosin beta 4, originally isolated from calf thymus [Low et al., Proc. Nat. Acad. Sci. USA 78, 1162-1166 (1981)] is present in a number of cell lines unrelated to the reticuloendothelium, including myoblasts and fibroblasts. It is also actively synthesized by these cell lines. Its content and rate of synthesis in the cell lines examined appear to be correlated with their ability to adhere and their motility.

Amino Acid Sequence↗
PubMed1983

Thymosin alpha 11: a peptide related to thymosin alpha 1 isolated from calf thymosin fraction 5.

Two peptides related to thymosin alpha 1 have been isolated from preparations of calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, is designated des-(25-28)-thymosin alpha 1. The other, named thymosin alpha 11, contains seven additional amino acid residues at the COOH terminus. The sequence of this peptide is: AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu- Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH. Thymosin alpha 11, in doses of less than 300 ng per mouse, protects susceptible inbred murine strains against opportunistic infections with Candida albicans. It is approximately equal to 30 times as potent as thymosin fraction 5 and approximately equal in potency to thymosin alpha 1.

Amino Acid Sequence↗
PubMed1983

Production and characterization of antibodies to thymosin beta 4.

Thymosin beta 4 antibodies have been raised in rabbits by conjugating the peptide to keyhole limpet hemocyanin, and a radioimmunoassay for thymosin beta 4 has been established utilizing tritiated thymosin beta 4 as binding ligand. RIA values for thymosin beta 4 in rat tissues reveal levels ranging between 8 and 448 micrograms/g wet weight. Highest levels are observed in spleen, although the peptide is also relatively abundant in thymus, lung, and nasal epithelium. Brain, kidney, liver and testis contain only 10 to 20% the concentration of thymosin beta 4 seen in spleen, whereas heart and muscle contain only 5 to 10% that of spleen. However, thymosin beta 4 probably has a ubiquitous distribution in the rat, being present in all tissues examined. The specificity of the antiserum used in the RIA has been defined. The thymosin beta 4 molecule seemingly bears two major epitopes that lie between amino acid residues 1-8 and 22-32. The antiserum does not cross-react with other unrelated peptides. The antiserum will precipitate biosynthetically labeled beta 4 from cultured rat peritoneal macrophage.

Amino Acid Sequence↗
PubMed1981

Factors that influence the translocation of the N-carboxybiotin moiety between the two sub-sites of pyruvate carboxylase.

The active site of pyruvate carboxylase, like those of all biotin-dependent carboxylases, is believed to consist of two spatially distinct sub-sites with biotin acting as a mobile carboxy-group carrier oscillating between the two sub-sites. Some of the factors that influence the location and rate of movement of the N-carboxybiotin were studied. The rate of carboxylation of the alternative substrate, 2-oxobutyrate, was measured at 0 degrees C in an assay system where the isolated enzyme--[14C]carboxybiotin was the carboxy-group donor. The results are consistent with the hypothesis that the location of the carboxybiotin in the active site is determined by the presence of Mg2+, acetyl-CoA and the oxo acid substrate. The presence of Mg2+ favours the holding of the complex at the first sub-site, whereas alpha-oxo acids induce the complex to move to the second sub-site. At low concentrations pyruvate induces this movement but does not efficiently act as a carboxy-group acceptor; hydroxypyruvate, glyoxylate and oxamate, though not carboxylated, still induce the movement. The allosteric activator acetyl-CoA exerts only a slight stimulation on the rate of translocation to the second sub-site, and this stimulation arises from an increase in the dissociation constant for Mg2+.

Acetoacetates↗