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J Segall

Publications and source records attributed to J Segall.

At least 19 recordsLinked to original sources
PubMed1992

The deduced sequence of the transcription factor TFIIIA from Saccharomyces cerevisiae reveals extensive divergence from Xenopus TFIIIA.

TFIIIA is an RNA polymerase III transcription factor that binds to the internal control region of the 5 S RNA gene as the first step in the assembly of a transcription complex. We have identified the gene encoding TFIIIA from Saccharomyces cerevisiae. Protein synthesized in vitro from the cloned gene has the same size, DNA-binding properties, and transcription factor activity as does purified yeast TFIIIA. Examination of the deduced sequence of the 50-kDa yeast transcription factor revealed the presence of nine zinc-finger motifs, a characteristic of Xenopus TFIIIA. Although the conservation of these nine putative DNA-binding domains is striking, the amino acid sequence throughout the corresponding fingers of the yeast and amphibian TFIIIAs has diverged extensively and in many instances the spacing between the residues that coordinate the zinc ions differs between the two proteins. A unique feature of the yeast protein is an 81-amino acid domain interrupting the repeated zinc-finger motifs between fingers 8 and 9. Additionally, the yeast and amphibian proteins differ in both the size and sequence of the amino- and carboxyl-terminal domains flanking the zinc fingers. The gene encoding yeast TFIIIA is present in single copy in the S. cerevisiae genome and is essential for cell viability. A carboxyl-terminal truncated form of the protein containing 4.5 zinc-finger motifs retains the ability to bind to DNA but is no longer active in promoting transcription in vitro.

Amino Acid Sequence↗
PubMed1991

Molecular analysis of amoeboid chemotaxis: parallel observations in amoeboid phagocytes and metastatic tumor cells.

Metastasis is a multistep process in which amoeboid chemotaxis plays a key role in the movement of tumor cells into and out of vessels. On a molecular level, much of what is known about amoeboid chemotaxis has been learned through work with Dictyostelium discoideum, a lower eukaryotic amoeboid phagocyte. One of the first and most crucial events to occur in the actin cytoskeleton following chemotactic stimulation is activation of actin nucleation. This is followed by incorporation of specific actin cross-linking proteins into the cytoskeleton, proteins which are implicated in the extension of pseudopods and filopods. Together, these events have been termed the Cortical Expansion Model for amoeboid chemotaxis. Detailed biochemical analysis has implicated a new actin-capping protein and has shown that one of the cross-linking proteins is Elongation Factor 1a, suggesting a link between chemotaxis and growth control. Preliminary data from parallel studies on neoplastic cells are presented.

Amino Acid Sequence↗
PubMed1990

Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae.

During sporulation of Saccharomyces cerevisiae, the four haploid nuclei generated by meiosis are encapsulated within multilayered spore walls. Taking advantage of the natural fluorescence imparted to yeast spores by the presence of a dityrosine-containing macromolecule in the spore wall, we identified and cloned two genes, termed DIT1 and DIT2, which are required for spore wall maturation. Mutation of these genes has no effect on the efficiency of spore formation or spore viability. The mutant spores, however, fail to accumulate the spore wall-specific dityrosine and lack the outermost layer of the spore wall. The absence of this cross-linked surface layer reduces the resistance of the spores to lytic enzymes, to ether, and to elevated temperature. Expression of the DIT and DIT2 genes is restricted to sporulating cells, with the DIT1 transcripts accumulating at the time of prospore enclosure and just prior to the time of dityrosine biosynthesis. Both genes act in a spore-autonomous manner implying that at least some of the activities responsible for forming the outermost layer of the spore wall reside within the developing spore rather than in the surrounding ascal cytoplasm. As the DIT2 gene product has significant homology with cytochrome P-450s, DIT2 may be responsible for catalyzing the oxidation of tyrosine residues in the formation of dityrosine.

Amino Acid Sequence↗
PubMed1989

Transcription of the 5 S rRNA gene of Saccharomyces cerevisiae requires a promoter element at +1 and a 14-base pair internal control region.

In vitro transcription of templates containing deletion-substitution mutations has localized two essential promoter elements of the 5 S rRNA gene of Saccharomyces cerevisiae. A promoter element spanning the start site of transcription extends from -14 to +8, and a short internal control region (ICR) extends from +81 to +94. Changes in spacing between these elements by more than a few base pairs significantly reduce transcription. The site of RNA polymerase III transcription factor A (TFIIIA) binding, mapped by determination of the G residues that are protected from methylation on exposure of the TFIIIA.5 S DNA complex to dimethyl sulfate, is coincident with the ICR. Incorporation of TFIIIC into the TFIIIA.5 S rRNA gene complex protects additional G residues 5' and 3' of the ICR from methylation.

Animals↗
PubMed1988

The SPS100 gene of Saccharomyces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation.

We previously described the use of a differential hybridization screen of a genomic DNA library of Saccharomyces cerevisiae to identify sporulation-specific (SPS) genes (A. Percival-Smith and J. Segall, Mol. Cell. Biol. 4:142-150, 1984). This initial screen identified 14 SPS genes that are first expressed 6 to 8 h after transfer of cells to sporulation medium. Accumulation of transcripts corresponding to these genes becomes maximal at 8 to 12 h of sporulation, the time at which meiotic events are nearing completion, and by 15 h of sporulation, transcript levels are beginning to decrease. In the present study two additional SPS genes, first expressed at 12 h of sporulation, were isolated. The steady-state level of transcripts corresponding to these two genes, termed SPS100 and SPS101, remains unchanged from 15 to 35 h, a time coincident with spore wall maturation. The nature of the putative 34.2-kilodalton protein encoded by the SPS100 gene is consistent with its being a component of the glycoprotein matrix of the spore wall; the protein contains a potential signal sequence and cleavage site and numerous sites for potential glycosylation. A MATa sps100/MAT alpha sps100 strain was found to be indistinguishable from the wild-type strain when assessed for efficiency of ascus formation and spore viability. However, a more detailed analysis of the mutant strain revealed that the SPS100 gene product serves a protective role during the early stages of spore wall formation. The time at which resistance to ether could first be detected in developing spores was delayed by 5 h in the mutant strain relative to the wild-type strain. This phenotype is presumably a reflection of a defect in spore wall maturation. This study has confirmed that temporally distinct classes of sporulation-specific genes are sequentially activated during the process of meiosis and spore formation and has shown that the SPS100 gene, identified on the basis of its developmental-specific expression pattern, contributes to spore development.

Amino Acid Sequence↗
PubMed1987

Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae.

We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta Pst) which could no longer inhibit sporulation. The SPS2 inserts of pAP290 and pAP290 delta Pst were found to direct equivalent levels of sporulation-specific transcription. We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype. We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.

Gene Amplification↗
PubMed1986

Assembly of a yeast 5 S RNA gene transcription complex.

The assembly of the yeast transcription factors IIIA, IIIB, and IIIC, represented by crude chromatographic fractions, into a stable complex on the 5 S RNA gene has been assessed using a template competition assay (Bogenhagen, D. F., Wormington, W. M., and Brown, D. D. (1982) Cell 28, 413-421). The yeast factor A can interact transiently with the yeast 5 S RNA gene, but both factors A and C are required for the formation of a stable complex. Factor A, but not factor C, is sequestered into this complex. Both factors A and C, however, are required for the sequestration of factor B into the 5 S RNA gene transcription complex. During this study, it was noted that an amount of factor C which was insufficient to lead to significant transcription of the 5 S RNA gene could nonetheless efficiently stabilize factor A on the template. This suggests that factor C may initially act catalytically to stabilize a transient interaction of factor A with the 5 S RNA gene before assuming a stoichiometric role in the assembly of the complete transcription complex. Alternative interpretations of this observation are discussed.

Genes, Fungal↗
PubMed1986

The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.

Amino Acid Sequence↗
PubMed1986

Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae.

A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

Amino Acid Sequence↗
PubMed1985

Characterization of factors and DNA sequences required for accurate transcription of the Saccharomyces cerevisiae 5 S RNA gene.

Cell-free extracts prepared from yeast cells have previously been shown to direct selective and accurate in vitro transcription of tRNA and 5 S RNA genes. We have further analyzed the transcription factors and DNA sequences required for in vitro transcription of the yeast 5 S RNA gene. Fractionation of a yeast extract has identified a 5 S RNA gene-specific factor required, in addition to the two previously described tRNA factors (Klekamp, M. S., and Weil, P. A. (1982) J. Biol. Chem. 257, 8432-8441), for accurate transcription of the 5 S RNA gene by RNA polymerase III. Transcription of variant 5 S RNA genes has indicated that a region of the gene extending from residue +57 to residue +99 is essential for directing specific initiation of transcription. Although the 5' flanking and initial coding sequence is not absolutely required for transcription of the gene, some of the variant genes which have substitutions in this region are less actively transcribed than the wild-type gene. Transcription initiates on some of the variant genes at a position equivalent to +1 in the substituted sequence, while on other variant genes transcription initiates further upstream.

Animals↗
PubMed1979

Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates.

The virus-associated (VA) RNAI gene in human adenovirus 2 DNA has been shown by Wu (Wu, G. J. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2175--2179) to be transcribed by RNA polymerase III in a human KB cell-free extract. In the present report we have examined the fidelity of transcription of adenovirus 2 DNA and Xenopus oocyte 5 S DNA templates by RNA polymerase III in extracts derived from cultured human, murine, and amphibian kidney cells, Size and sequences analysis of the discrete transcripts synthesized in these homologous and heterologous systems indicate that they result from accurate transcription of the corresponding genes. The specific transcripts identified include both the adenovirus VA RNAI and VA RNAII, Xenopus 5 S RNA, and VA RNAI and 5 S RNA species with elongated 3' termini. The extracts derived from the various cell types differ in the ability to discriminate between the two VA RNA genes or between the heterogeneous 5 S RNA genes in the cloned DNA fragment. Wherease the human cell extracts transcribe the VA RNAI and VA RNAII genes of adenovirus at a relative frequency close to that observed in isolated nuclei, the amphibian cell extract appears to transcribe only the VA RNAI gene. The amphibian cell extract transcribes primarily that 5 S RNA gene (within 5 S DNA) which encodes the dominant oocyte 5 S RNA, whereas the human cell extract transcribes at least two distinct 5 S RNA genes. Additionally, it is shown that the VA RNAI and VA RNAII genes have separate promotor sites. The kinetics of the transcription reactions have been examined and conditions optimal for specific transcription have been established by examining the effects of salt, metal ion, and template concentrations on both total and specific RNA synthesis. It is also shown that components in the cell-free extract (from human cells) are active in directing the accurate transcription of adenovirus DNA by purified RNA polymerase III.

Adenoviruses, Human↗
PubMed1979

Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA.

Transcription of Ad2 DNA templates in the presence of crude cellular extracts supplemented with exogenous (purified) RNA polymerase II is selectively and accurately initiated at the major late viral promoter at map position 16.45. Specific initiation has been demonstrated by a combination of hybridization, nuclease S1 mapping, size and partial sequence (fingerprint) analyses of the transcripts generated with various templates. With intact Ad2 DNA, transcription is terminated ell before the end of the 28 kb transcription unit is reached. With truncated templates (which contain intact promoter regions and several hundred base pair segments of the transcribed region) the expected run-off products are observed, along with a low level of prematurely terminated transcripts. The 560 nucleotide run-off product of the Sma l-f template (coordinates 11.6-18.2) was shown to contain all the large RNAase T1 oligonuc eotides that are characteristic of the corresponding in vivo transcript from this region; in addition, the 5 terminal undecanucleotide appears to be both capped and methylated. We have investigated various parameters (salt, metal ion and template concentrations) that affect the level of specific transcription in the crude system and have found that, under optimal conditions, specific transcription of Ad2 DNA continues for several hours. In addition, specific transcription initiation at the late promoter is observed with extracts derived from either virus-infected or uninfected KB cells and with class II RNA polymerases isolated from either human calf, murine or amphibian cells. RNA polymerase II from wheat germ does not function in this system.

Adenoviruses, Human↗
PubMed1977

Cloned Bacillus subtilis DNA containing a gene that is activated early during sporulation.

An endonuclease restriction fragment of Bacillus subtilis DNA has been identified that contains a gene whose transcription is activated early during the process of spore formation. This 4.4 kilobase (kb) DNA was detected by hybridizing electrophoretically separated Eco R1 restriction fragments with a radioactively labeled RNA of 0.4 kb from sporulating cells. The 4.4 kb B. subtilis DNA was then cloned and amplified in E. coli by insertion into the plasmid vector pMB9. Using the cloned B. subtilis DNA as a hybridization probe, we were able to detect the 0.4 kb transcript in total RNA from pulse-labeled bacteria. In wild-type cells, the gene coding for the 0.4 kb RNA was turned on within the first 30 min of spore formation. Although transcribed normally in a mutant blocked at stage II of spore development, the gene for the 0.4 kb RNA was not turned on in six different mutants blocked at stage 0 of sporulation. We conclude that the cloned B. subtilis DNA contains a gene whose transcription is regulated by events occurring at the onset of spore development.

Bacillus subtilis↗