Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph

Cells of several major algal groups are evolutionary chimeras of two radically different eukaryotic cells. Most of these “cells within cells” lost the nucleus of the former algal endosymbiont. But after hundreds of millions of years cryptomonads still retain the nucleus of their former red algal endosymbiont as a tiny relict organelle, the nucleomorph, which has three minute linear chromosomes, but their function and the nature of their ends have been unclear. We report extensive cryptomonad nucleomorph sequences (68.5 kb), from one end of each of the three chromosomes of Guillardia theta. Telomeres of the nucleomorph chromosomes differ dramatically from those of other eukaryotes, being repeats of the 23-mer sequence (AG)7AAG6A, not a typical hexamer (commonly TTAGGG). The subterminal regions comprising the rRNA cistrons and one protein-coding gene are exactly repeated at all three chromosome ends. Gene density (one per 0.8 kb) is the highest for any cellular genome. None of the 38 protein-coding genes has spliceosomal introns, in marked contrast to the chlorarachniophyte nucleomorph. Most identified nucleomorph genes are for gene expression or protein degradation; histone, tubulin, and putatively centrosomal ranbpm genes are probably important for chromosome segregation. No genes for primary or secondary metabolism have been found. Two of the three tRNA genes have introns, one in a hitherto undescribed location. Intergenic regions are exceptionally short; three genes transcribed by two different RNA polymerases overlap their neighbors. The reported sequences encode two essential chloroplast proteins, FtsZ and rubredoxin, thus explaining why cryptomonad nucleomorphs persist.

Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8m) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome.

An Escherichia coli strain with all chromosomal rRNA operons inactivated: Complete exchange of rRNA genes between bacteria

Current global phylogenies are built predominantly on rRNA sequences. However, an experimental system for studying the evolution of rRNA is not readily available, mainly because the rRNA genes are highly repeated in most experimental organisms. We have constructed an Escherichia coli strain in which all seven chromosomal rRNA operons are inactivated by deletions spanning the 16S and 23S coding regions. A single E. coli rRNA operon carried by a multicopy plasmid supplies 16S and 23S rRNA to the cell. By using this strain we have succeeded in creating microorganisms that contain only a foreign rRNA operon derived from either Salmonella typhimurium or Proteus vulgaris, microorganisms that have diverged from E. coli about 120–350 million years ago. We also were able to replace the E. coli rRNA operon with an E. coli/yeast hybrid one in which the GTPase center of E. coli 23S rRNA had been substituted by the corresponding domain from Saccharomyces cerevisiae. These results suggest that, contrary to common belief, coevolution of rRNA with many other components in the translational machinery may not completely preclude the horizontal transfer of rRNA genes.

Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B

The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation.

The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases

The hy1 mutants of Arabidopsis thaliana fail to make the phytochrome-chromophore phytochromobilin and therefore are deficient in a wide range of phytochrome-mediated responses. Because this defect can be rescued by feeding seedlings biliverdin IXα, it is likely that the mutations affect an enzyme that converts heme to this phytochromobilin intermediate. By a combination of positional cloning and candidate-gene isolation, we have identified the HY1 gene and found it to be related to cyanobacterial, algal, and animal heme oxygenases. Three independent alleles of hy1 contain DNA lesions within the HY1 coding region, and a genomic sequence spanning the HY1 locus complements the hy1–1 mutation. HY1 is a member of a gene family and is expressed in a variety of A. thaliana tissues. Based on its homology, we propose that HY1 encodes a higher-plant heme oxygenase, designated AtHO1, responsible for catalyzing the reaction that opens the tetrapyrrole ring of heme to generate biliverdin IXα.

CTXφ contains a hybrid genome derived from tandemly integrated elements

CTXφ is a filamentous, temperate bacteriophage whose genome includes ctxAB, the genes that encode cholera toxin. In toxigenic isolates of Vibrio cholerae, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5′ prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3′ element. The presence of tandem elements is required for production of virions: V. cholerae strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXφ, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXφ DNA into the V. cholerae chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXφ-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXφ does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXφ to new hosts.

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV1 and SV2 are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV1 is identical to that of human pituitary GHRH-R, whereas in SV2 exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV1 is identical to that of pituitary GHRH-R. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

Adaptive evolution of a candidate gene for aging in Drosophila

Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah (mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence at mth in population level samples of Drosophila melanogaster, D. simulans, and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.

Robust gut-specific gene expression in transgenic Aedes aegypti mosquitoes

Genetic modification of the vectorial capacity of mosquito vectors of human disease requires promoters capable of driving gene expression with appropriate tissue and stage specificity. We report on the characterization in transgenic Aedes aegypti of two mosquito gut-specific promoters. A 1.4-kb DNA fragment adjacent to the 5′ end of the coding region of the Ae. aegypti carboxypeptidase (AeCP) gene and a corresponding 3.4-kb DNA fragment at the 5′ end of the Anopheles gambiae carboxypeptidase (AgCP) gene were linked to a firefly luciferase reporter gene and introduced into the Ae. aegypti germ line by using Hermes and mariner (Mos1) transposons. Six independent transgenic lines were obtained with the AeCP construct and one with the AgCP construct. Luciferase mRNA and protein were abundantly expressed in the guts of transgenic mosquitoes in four of the six AeCP lines and in the AgCP line. Expression of the reporter gene was gut-specific and reached peak levels at about 24 h post-blood ingestion. The AeCP and AgCP promoters can be used to drive the expression of genes that hinder parasite development in the mosquito gut.

HCC-2, a human chemokine: Gene structure, expression pattern, and biological activity

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named “HCC-2.” The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKβ8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1γ, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1α. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.

Diversity and phylogeny of gephyrin: Tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins

Gephyrin is essential for both the postsynaptic localization of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (Moco) in different peripheral organs. Several alternatively spliced gephyrin transcripts have been identified in rat brain that differ in their 5′ coding regions. Here, we describe gephyrin splice variants that are differentially expressed in non-neuronal tissues and different regions of the adult mouse brain. Analysis of the murine gephyrin gene indicates a highly mosaic organization, with eight of its 29 exons corresponding to the alternatively spliced regions identified by cDNA sequencing. The N- and C-terminal domains of gephyrin encoded by exons 3–7 and 16–29, respectively, display sequence similarities to bacterial, invertebrate, and plant proteins involved in Moco biosynthesis, whereas the central exons 8, 13, and 14 encode motifs that may mediate oligomerization and tubulin binding. Our data are consistent with gephyrin having evolved from a Moco biosynthetic protein by insertion of protein interaction sequences.

New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study

Rheumatoid arthritis (RA), the most common autoimmune disease, is associated in families with other autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). Its genetic component has been suggested by familial aggregation (λs = 5), twin studies, and segregation analysis. HLA, which is the only susceptibility locus known, has been estimated to account for one-third of this component. The aim of this paper was to identify new RA loci. A genome scan was performed with 114 European Caucasian RA sib pairs from 97 nuclear families. Linkage was significant only for HLA (P < 2.5⋅10−5) and nominal for 19 markers in 14 other regions (P < 0.05). Four of the loci implicated in IDDM potentially overlap with these regions: the putative IDDM6, IDDM9, IDDM13, and DXS998 loci. The first two of these candidate regions, defined in the RA genome scan by the markers D18S68-D18S61-D18S469 (18q22–23) and D3S1267 (3q13), respectively, were studied in 194 additional RA sib pairs from 164 nuclear families. Support for linkage to chromosome 3 only was extended significantly (P = 0.002). The analysis of all 261 families provided a linkage evidence of P = 0.001 and suggested an interaction between this putative RA locus and HLA. This locus could account for 16% of the genetic component of RA. Candidate genes include those coding for CD80 and CD86, molecules involved in antigen-specific T cell recognition. In conclusion, this first genome scan in RA Caucasian families revealed 14 candidate regions, one of which was supported further by the study of a second set of families.

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl–tRNA synthetase in Arabidopsis thaliana

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl–tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl–tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNAMet genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl–tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.

Structure-based assignment of the biochemical function of a hypothetical protein: A test case of structural genomics

Many small bacterial, archaebacterial, and eukaryotic genomes have been sequenced, and the larger eukaryotic genomes are predicted to be completely sequenced within the next decade. In all genomes sequenced to date, a large portion of these organisms’ predicted protein coding regions encode polypeptides of unknown biochemical, biophysical, and/or cellular functions. Three-dimensional structures of these proteins may suggest biochemical or biophysical functions. Here we report the crystal structure of one such protein, MJ0577, from a hyperthermophile, Methanococcus jannaschii, at 1.7-Å resolution. The structure contains a bound ATP, suggesting MJ0577 is an ATPase or an ATP-mediated molecular switch, which we confirm by biochemical experiments. Furthermore, the structure reveals different ATP binding motifs that are shared among many homologous hypothetical proteins in this family. This result indicates that structure-based assignment of molecular function is a viable approach for the large-scale biochemical assignment of proteins and for discovering new motifs, a basic premise of structural genomics.