Proteins In Other Organisms Research Articles

Behavior of the [mi-3] mutation and conversion of polymorphic mtDNA markers in heterokaryons of Neurospora crassa.

We have examined the behavior of the [mi-3] mitochondrial mutation and two physical mtDNA markers in heterokaryotic cultures of Neurospora crassa. Previous workers showed that a 1.2-kilobase insertion in the larger polymorphic form of EcoRI-5 restriction fragment is a site of high frequency and rapid unidirectional gene conversion. We have confirmed this observation and determined by DNA sequence analysis that the insertion in the EcoRI-5 fragment corresponds precisely to an optional intron that contains a long open reading frame in the ND1 gene. Thus, the conversion of the short, intron-lacking, form of EcoRI-5 to the longer, intron-containing, form may be analogous to the unidirectional gene conversion events catalyzed by intron-encoded proteins in other organisms. The resolution of two polymorphic forms of the mtDNA EcoRI-9 restriction fragment in our heterokaryons differs from that observed previously and suggests that this locus is not a site of gene conversion in our heterokaryon pair. The size polymorphism of the EcoRI-9 fragments is due to a tandemly reiterated 78-base-pair sequence which occurs two times in the short form and three times in the long form. One copy of the repeat unit and 66 base pairs following it have been duplicated from the ND2 gene which is located about 30 kilobases distant on the mtDNA. In contrast to the [poky] mitochondrial mutant, which was completely dominant over wild-type mitochondria in heterokaryons, the [mi-3] mutant was recovered in only seven of twenty heterokaryons after ten cycles of conidiation and subculturing. The resolution of the [mi-3] or wild-type phenotype in heterokaryons may depend solely on random factors such as allele input frequency, drift, and segregation rather than specific dominant or suppressive effects.

Tetrins: polypeptides that form bundled filaments in Tetrahymena

The cortex of the ciliated protozoan Tetrahymena contains a number of fibrous elements, including a network of filaments that pervades the feeding organelle of this organism. The cluster of polypeptides (79-89K; K = 10(3) Mr) in Tetrahymena pyriformis GL-C that constitute these filaments has been purified by in vitro assembly after solubilization in 1.0 M KI. Four distinct sets of these polypeptides, designated 'tetrins', have been shown to be distinguishable from each other by immunochemical and biochemical criteria. The smallest filaments reassembled in vitro were 3-4 nm in diameter and these fine filaments were seen to be bundled together into thicker strands of varying diameters, similar to those within the cell. The thicker filament bundles were clearly distinguishable from intermediate filaments, but fine filaments in these bundles were superficially similar to the 2-5 nm filaments described as microtubule-associated proteins in other organisms. The ultrastructure of the tetrin filaments localized within the feeding organelle reveals a substantial presence of these filaments apart from microtubules. In addition, circular dichroism measurements indicate a relatively low alpha-helical content for these filaments and suggest that the tetrins may be substantially different from other fine filament proteins such as the tektins and giardins.

Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.

Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

Sequence and Regulation of a Gene Encoding a Human 89-Kilodalton Heat Shock Protein

Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

Nucleotide sequence analysis of soybean small heat shock protein genes belonging to two different multigene families

In soybean, the small heat shock proteins of 15 to 18 kDa are encoded in the nucleus by at least two different multigene families, designated class I and class VI. Genomic DNA sequences of two new heat shock genes and flanking regions were determined: Gmhsp18.5- C, a class I gene, and Gmhsp17.9- D, the first known class VI gene. Comparison of both genes revealed a moderate homology (approx. 38%) mainly within the 3′ ends of their coding regions. Hydropathic characterizations and secondary-structure predictions of the deduced amino acid sequences revealed two conserved domains within the C-terminal halves of the polypeptides that are also present in related proteins of other organisms. The transcription of both genes is heat shock dependent and the mRNA start sites, as determined by S 1 nuclease mapping, are located downstream from typical TATA box sequences and multiple heat shock promoter elements such as 5′ CT-GAA--TTC-AG. The putative promoter regions of the genes are preceded by long tracts of repetitive sequences with a high A + T content of 79 to 89%. which are bordered by runs of “simple sequences” such as (A) 12 13 , (T) 10 and (TA) 10. Similar characteristic features are present in the promoter and 5′-flanking regions of other soybean heat shock genes. The possible function of these distinct sequences is discussed.

The ParD‐ mutant of Escherichia coli also carries Aam mutation. The complete sequence of gyrA

The phenotype of a recently-described mutant (OV6), conditionally defective in chromosome partitioning and septal positioning, was originally thought to be due to a new gene (parD) mapping at 88.4 min. We have now shown that, in addition to the parD mutation, OV6 carries a gyrAam mutation and that this mutation is probably responsible for the gross phenotype of the mutant. We have cloned the gyrA gene, identified the GyrA protein, sequenced the gyrA gene and flanking genes, cloned and sequenced the gyrAam mutation, and identified its truncated product. In addition, we have identified the transcriptional start point of the gyrA gene. The E. coli GyrA protein has extensive homologies with Gyrase proteins of other organisms and weak sequence homologies with some eukaryotic cytoskeletal proteins.

Purification of Escherichia coli 30S ribosomal proteins by high performance liquid chromatography.

High performance liquid chromatography was applied to the separation of proteins derived from the Escherichia coli 30S ribosomal subunit. Several methods of separating this protein mixture has been tested: size-exclusion chromatography on hydrophilic phases; ion exchange and reversed phase chromatography (on C2 to C18 hydrocarbon-bonded supports). Various elution systems were examined in order to obtain pure proteins suitable for micro-sequence analysis. The resolution and yields of the proteins varied considerably, depending on the type of support and gradient system used. The best results were achieved with uniformly globular-shaped supports of large pore size, and by combining high performance size exclusion with rechromatography on reversed phase columns. Purification conditions for the individual proteins are listed. The methods employed avoid any precipitation step and allow easy identification of the proteins by one or two-dimensional gel electrophoresis, amino-acid analysis or direct manual or automatic micro-sequencing. Since the isolation time is much reduced compared with conventional purification procedures, the proteins obtained by the techniques described here are well suited for topographical and immunological studies or reconstitution assays. Ribosomal proteins of other organisms can be separated under similar conditions.

Cyclic AMP binding proteins in early embryos of Drosophila melanogaster

A variety of effects of cyclic AMP on cellular and subcellular phenomena suggest that there may be other modes of action of cyclic AMP than activation of protein kinase. It is also known that developing embryos contain cyclic AMP and its related enzymes. In order to explore the role of cyclic AMP in embryogenesis, a survey of proteins capable of binding cyclic AMP in the embryonic supernatant of Drosophila melanogaster was carried out. As the result, two cyclic AMP-binding proteins were found and characterized. The one (L) is, as expected, associated with protein kinase and has a dissociation constant of about 10 −9 M. Its molecular weight of 21 000 daltons is extremely small when compared with similar proteins in other organisms. The other (H), whose function is yet to be found, has a molecular weight of about 200 000 daltons and has a dissociation constant of about 10 −7 M. Some laxity in binding specificity of the latter protein among adenosine nucleotides was observed, but cyclic AMP is the strongest ligand among them.

Molecular characterization of the Drosophila genome.

A remarkable feature of evolution in the genus Drosophila is the conservation of the basic karyotype. Postulation of Robertsonian centric fusions (ROBERTSON 1916) is sufficient to explain most of the karyotypic rearrangements found in the various species. The fixation of large duplications of genetic material during the evolution of this genus appears to have been rare (STONE 1955). On the other hand, amino acid sequence data for proteins of other organisms indicate that many cistrons have very similar nucleotide sequences, and are presumably related by prior gene duplication (JUKES 1966). Physical studies of DNA of various eucaryotes also suggest that these genomes contain very large families of related cistrons (BRITTEN and KOHNE 1968). The size of such families is often in excess of that inferred from existing protein data (BRITTEN and KOHNE 1968; MCCARTHY 1967; WETMUR and DAVIDSON 1968). This may in some cases reflect the presence of cistrons with similar nucleotide sequences which specify proteins of overtly different functions. It is also likely that some of these families of genes have functions other than the determination of amino acid sequences. In keeping with the cytological studies, the reassociation kinetics of denatured Drosophila melanogaster DNA imply that the genome of this organism is much less complex, in terms of intragenome nucleotide sequence homologies, than those of vertebrates (LAIRD and MCCARTHY 1968b). Estimates made from these measurements suggest that perhaps 90% of D. melanogaster nucleotide sequences are not closely related to others elsewhere in the genome. In light of the extensive diversity within the genus Drosophila (LAIRD and MCCARTHY 1968a) , a detailed analysis of the intragenome homologies of representative species is expected to provide information about the evolution of genomes. In particular, our results indicate that genome simplicity extends to other Drosophila species and to a representative of another Dipteran family. In addition, there are variations among Diptera in genome size as inferred from the informational content of nucleotide sequences. MATERIALS AND METHODS Details of labeling and isolation of DNA and RNA from Drosophila, preparation of filterbound DNA, hybridization and duplex formation procedures, and analytical CsCl pycnography