Gene-specific expression of the actin multigene family of Dictyostelium discoideum

We have investigated the expression of 14 cloned genes of the 20-member actin multigene family of Dictyostelium discoideum using gene-specific mRNA complementary probes and an RNase protection assay. Actin gene expression was studied in vegetative cells and in cells at a number of developmental stages chosen to represent the known major shifts in actin mRNA and protein synthesis. At least 13 of these genes are expressed. A few genes are expressed very abundantly at 10% or more of total actin mRNA; however, the majority are maximally expressed at 1 to 5% of actin message. Although all of the genes are transcribed in vegetative cells, most genes appear to be independently regulated. Actin 8 appears to be transcribed at constant, high levels throughout growth and development. Actin 12 mRNA is maximally expressed in vegetative cells but the level is reduced appreciably by the earliest stage of development examined, while Actin 7 mRNA is specifically induced approximately sevenfold at this time. The rest of the genes appear to be induced 1.5 to 2-fold early in development, coincident with the increase in total actin mRNA. Since 12 of the genes code for extremely homologous proteins, it is possible that the large number of actin genes in Dictyostelium is utilized for precise regulation of the amount of actin produced at any stage of development, even though individual gene expression appears in some cases to be very stage-specific. In addition to these 13 actin genes, at least two and possibly four more genes are known to be expressed, because they are represented by complementary DNA clones, and an additional one or two expressed genes are indicated by primer extension experiments. Only one known gene, Actin 2-sub 2, is almost certainly a pseudogene. Thus the vast majority of Dictyostelium actin genes are expressed.     

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.     

A developmental study of the abnormal expression of alpha-cardiac and alpha-skeletal actins in the striated muscle of a mutant mouse

BALB/c mice possess a 5' duplication of the alpha-cardiac actin gene which is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue. This mutation therefore provides a potential tool for the study of the inter-relationship between the striated muscle actins. We have examined the expression of this actin gene pair throughout the development of skeletal and cardiac muscle in BALB/c mice. During embryonic and fetal development, the expression of these two genes is indistinguishable from that in normal mice, as determined by in situ hybridization. A quantitative postnatal study demonstrates that in the hearts of normal mice the level of alpha-cardiac actin mRNA declines, whereas that of alpha-skeletal actin increases. In mutant mice, these trends are exaggerated so that whereas normal mice have 95.8% alpha-cardiac mRNA and 4.2% alpha-skeletal mRNA in the adult heart, BALB/c mice have 52.4 and 47.6% of these mRNAs, respectively. This difference is also reflected at the protein level. In developing skeletal muscle, the expression of these genes follows kinetics similar to that observed in the heart with a decrease in the relative level of alpha-cardiac mRNA as the muscle matures. Cardiac actin mRNA levels are again lower in the mutant mouse, but here the effect is less striking because skeletal actin is the predominant isoform. These results are discussed in the context of the interaction between this actin gene pair in developing and adult striated muscle.     

Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications.

Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.     

Impaired flagellar regeneration due to uncoordinated expression of two divergent actin genes in Chlamydomonas

Chlamydomonas has two actin genes: one encoding a conventional actin (90% amino acid identity with mammalian actin) and the other a highly divergent actin (NAP; 64% identity). The expression of the two genes is regulated in a mutually exclusive manner. Thus, ida5, a mutant that lacks the conventional actin (CrA) gene, expresses NAP abundantly, whereas wild-type cells express NAP only negligibly under normal conditions. To explore the physiological significance of the two actins, chimeric genes with the 5' upstream region of one gene replaced by that of the other were constructed and used to transform ida5. The transformant (TF5) with a chimeric clone comprising the 5'-untranslated region from the NAP gene and the CrA-encoding sequence recovered the dyneins missing in ida5 and showed almost normal motility. After deflagellation of this transformant, however, only about 30% of cells grew flagella, unlike wild-type cells, >80% of which displayed reflagellation. Transformant TF10, which contains the CrA upstream region and NAP coding region, underwent reflagellation normally, as did the parent strain, ida5. In TF5, the mRNA level of both CrA and NAP was increased greatly during reflagellation. In light of the recent finding that NAP mRNA is expressed transiently upon reflagellation in wild-type cells, the described results suggest that 1) the expression of NAP mRNA is indispensable for flagellation and 2) robust expression of CrA may inhibit proper flagellation by interfering with the function of NAP in the early stages of reflagellation.     

Role of a membrane glycoprotein in Friend virus erythroleukemia: nucleotide sequences of nonleukemogenic mutant and spontaneous revertant viruses.

We previously isolated spontaneous env gene mutants of Friend spleen focus-forming virus that are nonleukemogenic in adult mice but form leukemogenic revertants in newborns; we found that the revertants contain secondary env mutations. To identify sites in the encoded membrane glycoprotein that are important for its pathogenic function, we molecularly cloned and partially sequenced the env genes of two mutant viruses (clone 63 and clone 4) and one revertant (clone 4REV). Clone 63 contained three noncontiguous point mutations that caused nonconservative amino acid substitutions of Gly-119----Arg-119, Cys-180----Tyr-180, and Gly-203----Arg-203 in the xenotropic-related domain of the env glycoprotein. These substitutions were presumably responsible for the altered electrophoretic and pathogenic properties of the mutant glycoprotein. The presence of these and several other G-A nucleotide substitutions at different sites in one spontaneous mutant provided striking evidence that error-rich proviruses can form during retroviral replication. Clone 4 contained a point mutation that generated a premature termination condon at amino acid residue 304 (Gln-304----Ochre-304). This termination codon was located immediately after the proposed xenotropic-ecotropic recombination site and eliminated the ecotropic-related domain, including the putative membrane anchor of the glycoprotein. Clone 4REV was a true revertant derived from clone 4 in which the premature termination codon had back-mutated to re-form the wild-type sequence. These results confirm an essential role for the env gene in Friend spleen focus-forming virus pathogenesis and suggest that the encoded membrane glycoprotein contains different domains that contribute to its pathogenic function.     

The Use of Restriction Fragment Length Polymorphisms and DNA Duplications to Study the Organization of the Actin Multigene Family in DICTYOSTELIUM DISCOIDEUM

The techniques of restriction fragment length polymorphism analysis and examination of gene copy number in duplication-bearing Dictyostelium discoideum strains have been used to map four actin genes of the wild-type strain NC4 to specific linkage groups. In part, this was accomplished by identification of restriction fragments corresponding to particular cloned actin genes using gene-specific probes from unique sequence 5' and 3' untranslated regions. Cloned gene Actin 8 (designation act-8) maps to linkage group I; Actins 12 (act-12) and M6 (actM6) to linkage group II. An uncloned gene (act-100) also maps to linkage group II in the same region as actM6, as defined by a chromosomal duplication. From analysis of other wild isolates of D. discoideum, it was determined that in these isolates at least two actin genes map to linkage group I and at least four map to linkage group II. These results demonstrate the utility of molecular techniques in genetic analysis of Dictyostelium, particularly for developmentally regulated genes that have been cloned but that have no identified mutant phenotypes.     

Isolation of actin genes in Bombyx mori: the coding sequence of a cytoplasmic actin gene expressed in the silk gland is interrupted by a single intron in an unusual position

To study the regulation of the gene(s) coding for the actin present in the microfilaments involved in the secretion of silk, we have probed a Bombyx mori genomic library with a Drosophila actin cDNA clone and selected 16 recombinant phages. They correspond to 3 different genomic fragments each containing a distinct actin coding sequence. Southern blots of genomic DNA probed with the cloned genes show that in Bombyx mori, there are at least 5 different actin genomic sequences. Two cloned genes A1 and A2 hybridize to a 1.7 kb long mRNA abundant in the carcass of the larva and thus probably code for muscle type actin. The third cloned gene, A3, hybridizes to two mRNAs of about 1.8 kb present in the silk gland and thus probably encodes a cytoplasmic actin. The coding sequence of this gene has been sequenced: it is almost identical to the Drosophila cytoplasmic actin genes but it has a single intron of 92 nucleotides within the codon 116, a position not observed in any other organism.     

Suppressors of snf2 Mutations Restore Invertase Derepression and Cause Temperature-Sensitive Lethality in Yeast

Mutations in the SNF2 gene of Saccharomyces cerevisiae prevent derepression of the SUC2 (invertase) gene, and other glucose-repressible genes, in response to glucose deprivation. We have isolated 25 partial phenotypic revertants of a snf2 mutant that are able to derepress secreted invertase. These revertants all carried suppressor mutations at a single locus, designated SSN20 (suppressor of snf2). Alleles with dominant, partially dominant and recessive suppressor phenotypes were recovered, but all were only partial suppressors of snf2, reversing the defect in invertase synthesis but not other defects. All alleles also caused recessive, temperature-sensitive lethality and a recessive defect in galactose utilization, regardless of the SNF2 genotype. No significant effect on SUC2 expression was detected in a wild-type (SNF2) genetic background. The ssn20 mutations also suppressed the defects in invertase derepression caused by snf5 and snf6 mutations, and selection for invertase-producing revertants of snf5 mutants yielded only additional ssn20 alleles. These findings suggest that the roles of the SNF2, SNF5 and SNF6 genes in regulation of SUC2 are functionally related and that SSN20 plays a role in expression of a variety of yeast genes.     

Escherichia coli K-12 lysyl-tRNA synthetase mutant with a novel reversion pattern

Fast-growing revertants have been selected from a slow-growing lysyl-tRNA synthetase mutant. All of the revertants had increased lysyl-tRNA synthetase activity compared with the mutant (5- to 85-fold), and in some revertants this amounted to two to three times the wild-type synthetase activity. Two-dimensional gel electrophoresis of a whole-cell extract of revertant IH2018 (1.5- to 2-fold wild-type synthetase activity) showed that the increase in synthetase activity is due to the induction of cryptic lysyl-tRNA synthetase forms and not to a change in the constitutive lysyl-tRNA synthetase. Genetic studies have shown that a locus termed rlu (for regulation of lysU ) which is cotransducible with purF at 49.5 min influences the amount of the cryptic lysyl-tRNA synthetase.     

Phenotypic reversion of an IS1-mediated deletion mutation: a combined role for point mutations and deletions in transposon evolution

We have physically characterised a deletion mutant of the R plasmid R100 which has lost all of the antibiotic resistances, including chloramphenicol resistance (Cmr), coded by its IS1-flanked r-determinant. The deletion was mediated by one of the flanking IS1 elements and terminates within the carboxyl terminus of the Cmr gene. DNA sequence analysis showed that the mutated gene would produce a protein 20 amino acids longer than the wild-type due to fusion with an open reading frame in the IS element. Surprisingly for a deletion mutation, rare, spontaneous Cmr revertants could be recovered. Two of the four revertants studied had frame shifts due to the insertion of a single AT base pair at the same position; the revertants would code for a protein five amino acids shorter than the wild-type. The other two revertants had acquired duplications of the 34-bp inverted terminal repeat sequences of the IS1 element and would direct the synthesis of a protein six amino acids longer than the wild-type. The reverted Cmr markers were still capable of transposition. These observations suggest a role for point mutations and small DNA rearrangements in the formation of new gene organisations produced by mobile genetic elements.     

Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203–215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon–intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages and that gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.