A novel yeast gene coding for a putative protein kinase

A yeast gene termed YKR2 coding for a putative protein kinase was isolated using the cloned cDNA for rabbit protein kinase C as a hybridization probe. The encoded protein YKR2, containing 677 amino acids, shows about 40% sequence identity in the kinase region to a family of serine/threonine-specific protein kinases from various species.     

The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not

The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.     

A novel second myostatin gene is present in teleost fish.

We report on the isolation and characterisation of the complete cDNA sequence encoding a novel bone morphogenetic protein-like protein (sbMSTN-b) in the teleost fish Sparus aurata. The encoded protein is 68% identical to S. aurata MSTN at the amino acid level, and homologues were also found in Umbrina cirrosa and Tetraodon nigroviridis. Phylogenetic analysis suggests that the MSTN-b gene may be present in most, perhaps all, teleost fish species. RT-PCR on different tissues/stages indicates that MSTN-b is expressed almost exclusively in the central nervous system, starting from late larval stages. Quantitative analyses indicate an increase of sbMSTN-b expression in the brain associated with metamorphosis, at the same time as completion of nervous system differentiation.     

Structure of a human smooth muscle actin gene (aortic type) with a unique intron site.

A recombinant phage containing an actin gene (lambda Ha201) was isolated from a human DNA library and the structure of the actin gene was determined. The amino acid sequences deduced from the nucleotide sequences of lambda Ha201 were compared with those of six actin isoforms; they matched those of bovine aortic smooth muscle actin, except for codon 309, which was valine (GTC) in lambda Ha201 and alanine (GCN) in bovine aortic smooth muscle actin. Southern blot hybridization experiments showed that the gene of normal human cells did not have the TaqI-sensitive site around position 309, whereas half of the genes of HUT14 cells did. These results indicate that one allele of the aortic smooth muscle actin gene in HUT14 cells has a transition point mutation (C----T) at codon 309 and that the amino acid sequences of normal human aorta and bovine smooth muscle actins are probably identical. In addition to the five introns interrupting exons at codons 150, 204, and 267, and between codons 41 and 42 and 327 and 328, which are common to skeletal muscle and cardiac muscle actin genes, the smooth muscle actin gene has two more intron sites between codons 84 and 85 and 121 and 122. The previously unreported intron site between codons 84 and 85 is unique to the smooth muscle actin gene. The intron site between codons 121 and 122 is common to beta-actin genes but is not found in other muscle actin genes. A hypothesis is proposed for the evolutionary pathway of the actin gene family.     

Molecular cloning and characterization of a putative protein kinase gene from the thermophilic fungus Thermomyces lanuginosus.

Based on the conserved amino acid sequence (DLKPEN) of serine-threonine protein kinase from several fungi, a degenerate primer was designed and synthesized. Total RNA was isolated from the thermophilic fungus Thermomyces lanuginosus. Using RACE-PCR, full-length cDNA of a putative serine-threonine protein kinase gene was cloned from T. lanuginosus. The full-length cDNA of T. lanuginosus protein kinase was 2551 bp and contained an 1806 bp open reading frame encoding a putative protein kinase precursor of 601 amino acid residues. Sequencing analysis showed that the cloned cDNA of T. lanuginosus had consensus protein kinase sequences. Conservative amino acid subdomains which most serine-threonine kinases contain can be found in the deduced amino acid sequence of T. lanuginosus putative protein kinase. Comparison results showed that the deduced amino acid sequence of T. lanuginosus putative protein kinase was highly homologous to that of Neurospora crassa dis1-suppressing protein kinase Dsk1. The putative protein kinase contained three arginine/serine-rich (SR) regions and two transmembrane domains. These showed that it might be a novel putative serine-threonine protein kinase.     

A novel selection system for recombinational and mutational events within an intron of a eucaryotic gene.

In order to identify a poison sequence that might be useful in studying illegitimate recombination of mammalian cell chromosomes, several DNA segments were tested for their ability to interfere with gene expression when placed in an intron. A tRNA gene and its flanking sequences (267 bp) were shown to inhibit SV40 plaque formation 100-fold, when inserted into the intron in the T-antigen gene. Similarly, when the same DNA segment was placed in the second intron of the adenosine phosphoribosyl transferase (APRT) gene from CHO cells, it inhibited transformation of APRT-CHO cells 500-fold. These two tests indicated that the 267-bp DNA segment contained a poison sequence. The poison sequence did not affect replication since the replication of poisoned SV40 genomes was complemented by viable SV40 genomes and poisoned APRT genes were stably integrated into cell chromosomes. Cleavage of the poison sequence in the SV40 T-antigen intron by restriction enzymes indicated that the tRNA structural sequences and the 5' flanking sequences were not required for inhibition of SV40 plaque formation. Sequence analysis of viable mutant SV40, which arose after transfection of poisoned genomes, localized the poison sequence to a 35 bp segment immediately 3' of the tRNA structural sequences.     

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata.

BACKGROUND: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (PIs) that are derived from a 43 kDa precursor protein, NaProPI. NaProPI contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native PIs. An unusual feature of NaProPI is that the structural domains lie across adjacent repeats and that the sixth PI domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPI repeat (aPI1) forms a basis for unravelling the mechanism by which this protein may have evolved. RESULTS: The three-dimensional structure of aPI1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native PIs. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPI, exists as a disordered loop in aPI1. The presence of this loop in aPI1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. CONCLUSIONS: A single repeat from NaProPI is capable of folding into a compact globular domain that displays native-like PI activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPI evolved.     

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

The psbF gene of chloroplast DNAs encodes the beta-subunit of cytochrome b-559 of the photosystem II reaction center. The psbF locus of Euglena gracilis chloroplast DNA has an unusual 1042 nt group II intron that appears to be formed from the insertion of one group II intron into structural domain V of a second group II intron. Using both direct primer extension cDNA sequencing and cDNA cloning and sequencing, we have determined that a 618 nt internal intron is first excised from the 1042 nt intron of psbF pre-mRNA, resulting in a partially spliced pre-mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. Therefore, the 1042 nt intron of psbF is a group II intron within another group II intron. We use the term 'twintron' to define this new type of genetic element. Intermediates in the splicing pathway were detected by northern hybridization. Splicing of both the internal and external introns occurs via lariat intermediates. Twintron splicing was found to proceed by a sequential pathway, the internal intron being removed prior to the excision of the external intron. A possible mechanism for twintron formation by intron transposition is discussed.     

Symbiotic origin of a novel actin gene in the cryptophyte Pyrenomonas helgolandii

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment; PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids, with this genome ultimately being lost when photosynthesis comes under full control of the "host" nucleus (e.g., as in heterokonts, haptophytes, and euglenophytes). Genes presently found in the nucleomorph seem to be restricted to those involved in its own maintenance and to that of the plastid; other genes were lost as the endosymbiont was progressively reduced to its present state. Surprisingly, we found that the cryptophyte Pyrenomonas helgolandii possesses a novel type of actin gene that originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage which are unrelated to plastid function. These genes are akin to the products of gene duplication or lateral transfer and provide a source of evolutionary novelty that can significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.     

Site-directed mutagenesis of the yeast actin gene: a test for actin function in vivo.

The yeast Saccharomyces cerevisiae has a single actin gene, ACT1, whose protein product is essential for cell viability. To study the structure-function relationship of this evolutionarily highly conserved protein, we have introduced into the gene several mutations leading to substitutions of amino acids that, by chemical crosslinking experiments, have previously been identified as potential sites for the interaction of actin with several actin-binding proteins and of actin monomers in filaments. The in vitro mutated actin genes were used to replace one chromosomal ACT1 allele in diploid cells. From diploid transformants, haploids that solely depended on mutant actins were isolated and their phenotypic alterations studied. The replacement of the N-terminal acidic residues (Asp2 and Glu4) with valine was functionally neutral. Substitutions of Asp11 led to dominant lethality. Substitutions of Lys191, Lys336, Trp356, Lys373 and Cys374 were without observable effect on cell growth, proliferation and morphology. Deletion of the C-terminal end, Lys-Cys-Phe-COOH, was lethal, whereas successive removal of the C-terminal Phe375 or Cys374 and Phe375 resulted in temperature sensitivity. At the nonpermissive temperature, the mutant cells were characterized by an increase in size, a tendency to lyse and significant alterations of the actin cytoskeleton.     

An mRNA-type intron is present in the Rhodotorula hasegawae U2 small nuclear RNA gene.

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.     

A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies

An extracellular, thermostable, alkaline lipase was partially purified from a thermophilic Bacillus strain J 33. It was optimally active at pH 8.0 at 60°C, retaining 50% activity at 70°C for 30 min. It had native molecular mass of 45 kDa. The lipase was stable in 90% (v/v) hexane or benzene mixtures in water. It converted 66% oleic acid at 0.25 M with 0.4 M methanol in hexane to methyl oleate at 60°C in 16 h. Activity was stimulated by Mg2 (10 mM) but inhibited by EDTA (10 mM) and PMSF (10 mM). It was stable in Triton X-100, Tween 20 and Tween 80 (0.1% v/v). © Rapid Science Ltd. 1998     

A very high fraction of unique intron positions in the intron-rich diatom Thalassiosira pseudonana indicates widespread intron gain

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.