ADrosophila homologue of theSchizosaccharomyces pombe act2 gene

Diverse proteins that are 35% to 55% identical to actins have been discovered recently in yeasts, nematodes, and vertebrates. In order to study these proteins systematically and relate their functions to those of conventional actins, we are isolating the corresponding genes from the genetically tractable eukaryote,Drosophila melanogaster. Here we report the isolation and partial characterization of aDrosophila homologue of theSchizosaccharomyces pombe act2 gene. Degenerate oligonucleotide primers specifying peptides that are highly conserved within the actin protein superfamily were used in conjunction with polymerase chain reaction (PCR) to amplify a portion of theDrosophila gene that we have namedactr66B. The corresponding full-length cDNA sequence encodes a protein of 418 residues that is 65% identical to the product of theS. pombe act2 gene, 80% identical to the bovineact2 homologue, but only 48% identical to the principalDrosophila cytoplasmic actin encoded by theAct5C actin gene. Alignment of the yeast, bovine, andDrosophila actin-related proteins shows that they have four peptide insertions, relative to conventional actins, three of which are well placed to modify actin polymerization and one that is likely to perturb the binding of myosin. Locations of two of the fiveactr66B introns are conserved betweenDrosophila and yeast genes, further attesting that they evolved from a common ancestor and are likely to encode proteins having similar functions. We demonstrate that theDrosophila gene is located on the left arm of chromosome 3, within subdivision 66B. Finally, we show by RNA blot-hybridization that the gene is expressed at low levels, relative to conventional nonmuscle actin, in all developmental stages. From these and other observations we infer that the actr66B protein is a minor component of all cells, perhaps serving to modify the polymerization, structure, and dynamic behavior of actin filaments. Our work was supported by grants from the NIH and the Muscular Dystrophy Association to E.A.F. Sequences described herein have been filed in the GenBank Database under Accession Number X71789.     

Characterization of the calmodulin gene family in wheat: structure, chromosomal location, and evolutionary aspects

Calmodulin is a ubiquitous transducer of calcium signals in eukaryotes. In diploid plant species, several isoforms of calmodulin have been described. Here, we report on the isolation and characterization of calmodulin cDNAs corresponding to 10 genes from hexaploid (bread) wheat (Triticum aestivum). These genes encode three distinct calmodulin isoforms; one isoform is novel in that it lacks a conserved calcium binding site. Based on their nucleotide sequences, the 10 cDNAs were classified into four subfamilies. Using subfamily-specific DNA probes, calmodulin genes were identified and the chromosomal location of each subfamily was determined by Southern analysis of selected aneuploid lines. The data suggest that hexaploid wheat possesses at least 13 calmodulin-related genes. Subfamilies 1 and 2 were both localized to the short arms of homoeologous-group 3 chromosomes; subfamily 2 is located on all three homoeologous short arms (3AS, 3BS and 3DS), whereas subfamily 1 is located only on 3AS and 3BS but not on 3DS. Further analysis revealed thatAegilops tauschii, the presumed diploid donor of the D-genome of hexaploid wheat, lacks a subfamily-1 calmodulin gene homologue, whereas diploid species related to the progenitors of the A and B genomes do contain such genes. Subfamily 3 was localized to the short arm of homoeologous chromosomes 2A, 2B and 2D, and subfamily 4 was mapped to the proximal regions of 4AS, 4BL and 4DL. These findings suggest that the calmodulin genes within each subfamily in hexaploid wheat represent homoeoallelic loci. Furthermore, they also suggest that calmodulin genes diversified into subfamilies before speciation ofTriticum andAegilops diploid species.     

FXY2/MID2, a Gene Related to the X-Linked Opitz Syndrome Gene FXY/MID1, Maps to Xq22 and Encodes a FNIII Domain-Containing Protein That Associates with Microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

FXY2/MID2, a gene related to the X-linked Opitz syndrome gene FXY/MID1, maps to Xq22 and encodes a FNIII domain-containing protein that associates with microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.     

The Asc locus for resistance to Alternaria stem canker in tomato does not encode the enzyme aspartate carbamoyltransferase

The fungal disease resistance locus Alternaria stem canker (Asc) in tomato has been suggested to encode the enzyme aspartate carbamoyltransferase (AC Tase). To test this hypothesis a segment of the tomato ACTase gene was amplified by the polymerase chain reaction (PCR) using degenerate primers. The PCR product obtained was subsequently used to isolate an ACTase cDNA clone. Restriction fragment length polymorphism (RFLP) linkage analysis showed that the ACTase gene and the Asc locus do not cosegregate. RFLP mapping positioned the ACTase gene on chromosome 11, while the Asc locus is located on chromosome 3. These results exclude the possibility that the ACTase protein is encoded by the Asc locus.     

Organization of the genetic locus for chicken myosin light chain kinase is complex: Multiple proteins are encoded and exhibit differential expression and localization

We report that the genetic locus that encodes vertebrate smooth muscle and nonmuscle myosin light chain kinase (MLCK) and kinase-related protein (KRP) has a complex arrangement and a complex pattern of expression. Three proteins are encoded by 31 exons that have only one variation, that of the first exon of KRP, and the genomic locus spans approximately 100 kb of DNA. The three proteins can differ in their relative abundance and localization among tissues and with development. MLCK is a calmodulin (CaM) regulated protein kinase that phosphorylates the light chain of myosin II. The chicken has two MLCK isoforms encoded by the MLCK/KRP locus. KRP does not bind CaM and is not a protein kinase. However, KRP binds to and regulates the structure of myosin II. Thus, KRP and MLCK have the same subcellular target, the myosin II molecular motor system. We examined the tissue and cellular localization of KRP and MLCK in the chicken embryo and in adult chicken tissues. We report on the selective localization of KRP and MLCK among and within tissues and on a differential distribution of the proteins between embryonic and adult tissues. The results fill a void in our knowledge about the organization of the MLCK/KRP genetic locus, which appears to be a late evolving regulatory paradigm, and suggest an independent and complex regulation of expression of the gene products from the MLCK/KRP genetic locus that may reflect a basic principle found in other eukaryotic gene clusters that encode functionally linked proteins. J. Cell. Biochem. 70:402–413, 1998. © 1998 Wiley-Liss, Inc.     

Molecular genetics of Drosophila alpha-actinin: mutant alleles disrupt Z disc integrity and muscle insertions

We have isolated a Drosophila melanogaster alpha-actinin gene and partially characterized several mutant alleles. The Drosophila protein sequence is very similar (68% identity) to those of chicken alpha-actinin isoforms, but less closely related (30% identity) to Dictyostelium alpha-actinin. The gene is within subdivision 2C of the X chromosome, coincident with 15 lethal (1)2Cb mutations. At least four alleles, l(1)2Cb1, l(1)2Cb2, l(1)2Cb4, and l(1)2Cb5 are interrupted by rearrangement breakpoints and must be null. In all four cases, hemizygous mutants complete embryogenesis and do not die until the second day of larval growth, signifying that either the role of alpha-actinin in nonmuscle cells is redundant or that a distinct and only distantly related gene encodes the non-muscle isoform. Allelic but less severely affected fliA mutants are apparently due to point mutations, and develop into adults having thoracic muscle abnormalities. EM of mutant muscles reveals that Z discs and myofibrillar attachments are disrupted, whereas epithelial "tendon" cells are less affected. We discuss these phenotypes in the light of presumed in vivo alpha-actinin functions.     

Isolation of aDrosophila gene encoding glutathioneS-transferase

We have isolated a Drosophila gene, DmGST-2, that encodes glutathione S-transferase, a homo- or heterodimeric enzyme thought to be involved in detoxification of xenobiotics, including known carcinogens. The encoded protein has a primary sequence that is more similar to mammalian placental and nematode GSTs than that of a previously described Drosophila GST gene, herein referred to as DmGST-1. We provide a physical map of the gene and show that it specifies at least two mRNAs, measuring 1.9 and 1.6 kb, which differ only in the lengths of their 3' untranslated regions. Both of the mRNAs are present during all developmental stages. In situ hybridization of the DmGST-2 gene to larval polytene chromosomes places it within the 53F subdivision of chromosome 2, and Southern blotting to chromosomal DNA indicates that the gene has no close relatives within the Drosophila genome. Our results make possible molecular genetic approaches for further elaborating the function of glutathione S-transferases in insect development and physiology, in the metabolism of plant toxins, and in conferring insecticide resistance.     

Alternative intronic promoters in development and disease

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.     

The secY-rpmJ region of the spc ribosomal protein operon in Escherichia coli: structural alterations affecting secY expression

Summary A unique feature of the spc ribosomal protein operon is that its region distal to the promoter contains a gene (secY) for an integral membrane protein, followed by an open reading frame termed X which has recently been proposed to encode a new ribosomal protein (protein B). We now show that the open reading frame X indeed directs the synthesis of a protein with electrophoretic mobilities similar to the B protein, and this supports the proposal that X may be more appropriately called rpmJ. Insertion of a plasmid sequence into the secY-rpmJ boundary of the chromosome caused a reduced expression of secY probably by destabilizing the secY part of the message. The results of complementation experiments suggested that a normal level of expression of rpmJ is not required for growth or protein secretion.     

Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana

The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 andsdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.     

Two genes encoding gas vacuole proteins in Halobacterium halobium

Summary The archaebacterium Halobacterium halobium contains two related gas vacuole protein-encoding genes (vac). One of these genes encodes a protein of 76 amino acids and resides on the major plasmid. The second gene is located on the chromosome in a (G+C)-rich DNA fraction and encodes a slightly larger but highly homologous protein consisting of 79 amino acids. The plasmid encoded vac gene is transcribed constitutively throughout the growth cycle while the chromosomal vac gene is expressed during the stationary phase of growth. Comparison of the nucleotide sequences of the two genes indicates differences in the putative promoter regions as well as 35 single base-pair exchanges within the coding regions of the two genes. The majority of the nucleotide exchanges in the coding region occur in the third position of a codon triplet generating the codon synonym. The only differences between the two encoded proteins are the exchange of 2 amino acids (positions 8 and 29) and a deletion of 3 amino acids near the carboxy-terminus of the plasmid encoded vac protein. The genomic DNAs from other halobacterial isolates (Halobacterium sp. SB3, GN101 and YC819-9) were found to contain only a chromosomal vac gene copy. There is a high conservation of the chromosomal vac gene and the genomic region surrounding it among the halobacterial strains investigated.     

Trifluoperazine inhibition of contraction in permeabilized skeletal, cardiac and smooth muscles

To gain insights into the mechanism of the central helix of calmodulin and troponin-C in the Ca2(+)-regulation of force development in striated and smooth muscles, the present study was made of the TFP induced inhibition of contraction, and of the uptake of these proteins by skinned fibers. Calmodulin was four-fold more sensitive to TFP than TnC, but the inhibition was found to be identical for skeletal and cardiac muscles despite the differences in their troponin-C isoforms. Also, the results were comparable between fast-twitch fiber, when calmodulin was exchanged for troponin-C to act on TnI, and smooth muscle, where calmodulin acts on myosin light chain kinase. These findings indicate that the inhibition of force by TFP is entirely due to its binding to the hydrophobic sites in the central helix. The uptakes of troponin-C and calmodulin were also different, and this is explained by a TFP-independent domain in troponin-C that binds TnI.