Expression of functional Xenopus TFIIIA in Escherichia coli.
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Biomedical subjects
L J Korn
Publications and source records attributed to L J Korn.
Upstream sequences required for transcription of the TFIIIA gene in Xenopus oocytes.
The DNA sequences required for efficient initiation of transcription of the Xenopus transcription factor IIIA (TFIIIA) gene were determined by microinjecting a series of deletion and linker substitution mutants into Xenopus oocyte nuclei. An upstream activating sequence, which resides between residues -283 and -238, and perhaps a second sequence between -167 and -122 preceding the transcription start site, together stimulate transcription about 30-fold. The distance between the two sequences and the distance from them to the initiation site can vary by at least 13 base pairs without loss of activity. The TFIIIA upstream sequences can stimulate transcription of other genes, for example, they stimulate transcription from the herpes thymidine kinase promoter about 30-fold.
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Nuclear factors in B lymphoma enhance splicing of mouse membrane-bound mu mRNA in Xenopus oocytes.
Regulation of the synthesis of membrane-bound and secreted immunoglobulin mu heavy chains at the level of RNA processing is an important element for B cell development. The precursor mu RNA is either polyadenylated at the upstream poly(A) site (for the secreted form) or spliced (for the membrane-bound form) in a mutually exclusive manner. When the mouse mu gene linked to the SV40/HSV-TK hybrid promoter was microinjected into Xenopus oocytes, the mu messenger RNA (mRNA) was altered by coinjection of nuclei of mouse surface IgM-bearing B-lymphoma cells to include the synthesis of the membrane-bound form. An increase in the membrane-bound form was not observed when nuclei of IgM-secreting hybridoma cells or fibroblast cells were coinjected. Deletion of the upstream poly(A) site did not eliminate the effect of B-lymphoma nuclei suggesting that membrane-specific splicing is stimulated. Further, splicing of other mu gene introns was not affected by coinjection of B-lymphoma nuclei. These results suggest that mature B cells contain one or more transacting nuclear factors that stimulate splicing specific for membrane-bound mu mRNA.
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Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme.
Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.
Bacterial Proteins↗
Regulation of differential processing of mouse immunoglobulin mu heavy-chain mRNA.
The switch between the synthesis of membrane-bound and secreted IgM during B cell differentiation is accomplished by producing, from a single gene, two alternative forms of mu heavy-chain mRNA that differ only in their 3' termini. The precursor mu RNA is either polyadenylated at the first poly(A) site, for secreted mu mRNA, or spliced between the C4 and M1 exons, for membrane-bound mu mRNA, in a mutually exclusive manner. To elucidate the molecular mechanism of the differential processing of mouse mu mRNA, we analyzed the expression of various mouse mu gene constructs stably transfected into mouse cell lines. In B cell lines, processing of the exogenously transfected mu gene transcripts accurately reflected the developmental stage of the recipient cells: both secreted and membrane-bound mu mRNAs are produced in early-stage B cells while secreted mu mRNA is primarily produced in late-stage B cells. In fibroblast cell lines, mu mRNAs transcribed from the Moloney murine sarcoma virus LTR promoter were processed primarily to the secreted form. Thus, production of the secreted form seems to be the non-regulated processing pattern. When the splicing signal of the C4-M1 intron was mutagenized, polyadenylation at the first poly(A) site occurred efficiently regardless of the recipient cell lines. On the other hand, when the polyadenylation signal was mutagenized, the splicing occurred efficiently in early-stage B cells, but only weakly in late-stage B cells and fibroblast cells. These results suggest that the splicing of the C4-M1 intron is stimulated in early-stage B cells.
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Ras p21 as a potential mediator of insulin action in Xenopus oocytes.
The oncogene protein product (p21) of the ras gene has been implicated in mediating the effects of a variety of growth factors and hormones. Microinjection of monoclonal antibody 6B7, which is directed against a synthetic peptide corresponding to a highly conserved region of p21 (amino acids 29 to 44) required for p21 function, specifically inhibited Xenopus oocyte maturation induced by incubation with insulin. The inhibition was dose-dependent and specific since (i) the same antibody had no effect on progesterone-induced maturation, (ii) immunoprecipitation and Western blotting indicated that the antibody recognized a single protein of molecular weight 21,000 in oocyte extracts, and (iii) inhibition was not observed with identical concentrations of normal immunoglobulin. Thus, p21 appears to be involved in mediating insulin-induced maturation of Xenopus oocytes. Furthermore, the mechanism may involve phosphorylation of p21, as p21 was found to be a substrate of the insulin receptor kinase.
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Effects of intron length on differential processing of mouse mu heavy-chain mRNA.
Production of membrane-bound and secreted forms of mouse mu heavy-chain mRNA is controlled by differential processing in a developmental-stage-specific manner. We have analyzed the effects of various deletions and insertions in the C4-M1 intron of the mouse mu gene on the differential processing of mu mRNA. We show that there is a correlation between the length of the C4-M1 intron and the molar ratio of membrane-bound to secreted mu mRNAs, i.e., the shorter the C4-M1 intron, the higher the ratio. Since the poly(A) addition signal in the C4-M1 intron seems to be intact in the mutant mu genes, it is likely that the efficiency of splicing of the C4-M1 intron is affected by changes in the intron length.
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Structure of the gene for Xenopus transcription factor TFIIIA.
The eucaryotic transcription factor TFIIIA is required for 5S RNA transcription in Xenopus, and changes in the level of TFIIIA have been implicated in the differential expression of 5S RNA genes. In this paper, we report the isolation and sequencing of the X. laevis TFIIIA gene. The gene is approximately 11 kb in length and consists of 9 coding segments separated by 8 introns. A sequence of 30 amino acid residues is known to repeat imperfectly 9 times in tandem within the TFIIIA protein, and Miller et al. (EMBO J. 4, 1609-1614, 1985) proposed that TFIIIA evolved by duplication of a primordial 30 amino acid residue unit. Our results from DNA sequence analysis support their proposal by showing that some of the exon-intron boundaries correspond closely to the repeating unit. We also found that the 5' flanking sequence of the TFIIIA gene contains a TATA box (TATATAA) at position -32 and a CAAT box (GCCAATCC) at position -96 and that the site of polyadenylation is 255 residues 3' of the stop codon. Finally, we have shown that the coding sequence of the TFIIIA gene is significantly polymorphic.
Amino Acid Sequence↗
Early replication and expression of oocyte-type 5S RNA genes in a Xenopus somatic cell line carrying a translocation.
In Xenopus somatic cells, the somatic-type 5S RNA genes replicate early in S phase, bind the transcription factor TFIIIA, and are expressed; in contrast, the late replicating oocyte-type genes do not bind TFIIIA and are transcriptionally inactive. These facts support a model in which the order of replication of the somatic-type versus the oocyte-type 5S genes causes their differential expression in somatic cells due to sequestration of TFIIIA by the early-replicating somatic genes. Here we provide further evidence for the model by showing that in one Xenopus cell line in which some oocyte-type 5S genes are translocated, some oocyte-type 5S genes replicate early and are expressed.
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Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase.
Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor beta subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor beta subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.
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Differential order of replication of Xenopus laevis 5S RNA genes.
In Xenopus laevis there are two multigene families of 5S RNA genes: the oocyte-type 5S RNA genes which are expressed only in oocytes and the somatic-type 5S RNA genes which are expressed throughout development. The Xenopus 5S RNA replication-expression model of Gottesfeld and Bloomer (Cell 28:781-791, 1982) and Wormington et al. (Cold Spring Harbor Symp. Quant. Biol. 47:879-884, 1983) predicts that the somatic-type 5S RNA genes replicate earlier in the cell cycle than do the oocyte-type genes. Hence, the somatic-type 5S RNA genes have a competitive advantage in binding the transcription factor TFIIIA in somatic cells and are thereby expressed to the exclusion of the oocyte-type genes. To test the replication-expression model, we determined the order of replication of the oocyte- and somatic-type 5S RNA genes. Xenopus cells were labeled with bromodeoxyuridine, stained for DNA content, and then sorted into fractions of S phase by using a fluorescence-activated cell sorter. The newly replicated DNA containing bromodeoxyuridine was separated from the lighter, unreplicated DNA by equilibrium centrifugation and was hybridized with DNA probes specific for the oocyte- and somatic-type 5S RNA genes. In this way we found that the somatic-type 5S RNA genes replicate early in S phase, whereas the oocyte-type 5S RNA genes replicate late in S phase, demonstrating a key aspect of the replication-expression model.
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A comprehensive sequence analysis program for the IBM personal computer.
We have developed a versatile program for the analysis of nucleic acid and protein sequences on the IBM Personal Computer. The program is interactive and self-instructing. It contains all the features generally found in sequence analysis programs on large computers, including extensive homology routines, as well as new procedures for the entry of sequence data. The program contains facilities to store and utilize the entire Nucleic Acid Sequence Data Bank. We have devised a new algorithm to find restriction enzyme sites, which allows our microcomputer program to find all sites on a small plasmid for 100 different enzymes in 1 to 2 minutes.
Amino Acid Sequence↗
Analysis of biological sequences on small computers.
We review a wide range of programs for micro- and minicomputers that facilitate the collection and analysis of DNA, RNA, or protein sequences. Special-purpose programs that perform a single function and general-purpose programs that perform a set of functions are both considered. The information presented should be useful to any biologist who wishes to obtain a computer program to aid in sequence investigations.
Amino Acid Sequence↗
Comparative analysis of human and bovine papillomaviruses.
A method is presented for the analysis and comparison of nucleic acid and protein sequences utilizing all identity blocks (the term "identity block" refers to a set of consecutive matches between two sequences) above a prescribed length. Moreover, such identity blocks are determined for various groupings of amino acids according to chemical, functional, charge, and hydrophobic classifications. Alignment maps based on these classifications and containing all statistically significant identity blocks between two or more sequences are constructed. New theoretical results for determining the expected length of the longest identity block between sequences are also presented and are used, along with permutation procedures, to ascertain the significance of sequence identity blocks. As an example of the type of information that can be obtained, comparison has been made of the complete DNA sequences and the E1, E2, L1, and L2 genes of human and bovine papillomaviruses based on the classification schemes described above.
Amino Acids↗
Chromosomal mapping of Xenopus 5S genes: somatic-type versus oocyte-type.
Xenopus 5S RNA genes exhibit a pattern of differential expression during development in which some members (oocyte-type) are transcribed only in oocytes, while others (somatic-type) are expressed in both oocytes and somatic cells. Using cloned DNA probes specific for each gene type, we determined the positions of these genes on Xenopus metaphase chromosomes by in situ hybridization. Somatic-type 5S genes in both X. laevis and X. borealis are located at the distal end of the long arm of only one chromosome (number 9). The oocyte-type 5S RNA genes are found at the distal ends of the long arms of most Xenopus chromosomes, including chromosome 9. Thus, large scale differences in chromosomal location cannot explain the selective expression of these genes, as suggested previously.
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New approaches for computer analysis of nucleic acid sequences.
A new high-speed computer algorithm is outlined that ascertains within and between nucleic acid and protein sequences all direct repeats, dyad symmetries, and other structural relationships. Large repeats, repeats of high frequency, dyad symmetries of specified stem length and loop distance, and their distributions are determined. Significance of homologies is assessed by a hierarchy of permutation procedures. Applications are made to papovaviruses, the human papillomavirus HPV, lambda phage, the human and mouse mitochondrial genomes, and the human and mouse immunoglobulin kappa-chain genes.
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Oocyte extracts reactivate developmentally inert Xenopus 5S genes in somatic nuclei.
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Developmental inactivity of 5S RNA genes persists when chromosomes are cut between genes.
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