Generation of Minute phenotypes by a transformed antisense ribosomal protein gene

Antisense RNAs have been used for gene interference experiments in many cell types and organisms. However, relatively few experiments have been conducted with antisense genes integrated into the germ line. In Drosophila reduced ribosomal protein (r-protein) gene function has been hypothesized to result in a Minute phenotype. In this report we examine the effects of antisense r-protein 49 expression, a gene known to correspond to a Minute mutation An antisense rp49 gene driven by a strong and inducible promoter was transformed into the Drosophila germ line. Induction of this gene led to the development of flies with weak Minute phenotypes and to the transient arrest of oogenesis. Parameters that may affect the success of antisense gene inactivation are discussed. © 1992 Wiley-Liss, Inc.     

Ribosomal protein S14 is not responsible for the Minute phenotype associated with the M(1)7C locus in Drosophila melanogaster.

Summary A locus associated with a severe Minute effect has been mapped at 7C on the X chromosome of Drosophila melanogaster. Previous work has suggested that this Minute encodes ribosomal proteins S14A and S141B. We have made a chromosomal deficiency that removes the S14 ribosomal protein genes, yet does not display the Minute phenotype. These data suggest that the S14 genes do not actually correspond to the Minute locus.     

A newly identifiedMinute locus,M(2)32D, encodes the ribosomal protein L9 inDrosophila melanogaster

A gene encoding a ubiquitously expressed mRNA inDrosophila melanogaster was isolated and identified as the gene for ribosomal protein L9 (rpL9) by its extensive sequence homology to the corresponding gene from rat. TherpL9 gene is localized in polytene region 32D where two independent P element insertions flanking the locus are available. Remobilization of either P element generated lines with a typicalMinute phenotype, e.g. thin and short bristles, prolonged development, and female semisterility in heterozygotes as well as homozygous lethality. All these characteristics can be rescued when a 3.9 kb restriction fragment containing therpL9 gene is reintroduced by P element-mediated germline transformation. This result confirms thatM(2)32D codes for ribosomal protein L9.     

Mapping the nucleolus organizer region,seed protein loci and isozyme loci on chromosome 1R in rye

Summary The nucleolus organizer region located on the short arm of chromosome 1R of rye consists of a large cluster of genes that code for ribosomal RNA (designated the Nor-R1 locus). The genes in the cluster are separated by spacer regions which can vary in length in different rye lines. Differences in the spacer regions were scored in two families of F₂ progeny. Segregation also occurred, in one or both of the families, at two seed protein loci and at two isozyme loci also located on chromosome 1R. The seed protein loci were identified as the Sec 1 locus controlling -secalins located on the short arm of chromosome 1R and the Sec 3 locus controlling high-molecular-weight secalins located on the long arm of 1R. The two isozyme loci were the Gpi-R1 locus controlling glucose-phosphate isomerase isozymes and the Pgd 2 locus controlling phosphogluconate dehydrogenase isozymes. The data indicated linkage between all five loci and map distances were calculated. The results indicate a gene order: Pgd 2 ... Sec 3 ... [centromere] ... Nor-R1 ... Gpi-R1 ... Sec 1. Evidence was obtained that rye possesses a minor 5S RNA locus (chromosome location unknown) in addition to the major 5S RNA locus previously shown to be located on the short arm of chromosome 1R.     

Identification and characterization of the gene for Drosophila L3 ribosomal protein

A cDNA clone that encodes a Drosophila homologue of ribosomal protein L3 was isolated from a Drosophila ovary gridded cDNA library. The Drosophila ribosomal protein L3 gene (RpL3) is highly conserved with ribosomal protein L3 genes in other organisms. It is a single copy gene and maps to position 86D5–10 on polytene chromosomes. A Minute gene in this region, M(3)86D, is a possible candidate to encode RPL3. RPL3 message is expressed ubiquitously. A partial RPL8 cDNA clone was also isolated and mapped to 62F.     

The ribosomal protein genes and Minute loci of Drosophila melanogaster

Background

Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes.

Results

We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci.

Conclusion

This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome.     

Search for Drosophila genes based on patterned expression of mini-white reporter gene of a P lacW vector in adult eyes

Developmental expression of transduced mini-white(w) gene of Drosophila is sensitive to its flanking genomic enhancers. Taking advantage of this phenomenon, we mobilized a P lacW transposon and screened for new transposant lines which showed patterned expression of the mini-w gene in adult eyes. From a screen of about 1,000 independent P lacW transposant lines on the second chromosome, we identified 7 lines which showed patterned w expression in adult eyes. These P insertions were assigned to engrailed, wingless and teashirt genes based on their chromosomal locations, developmental expression of the lacZ reporter gene, lethal embryonic mutant phenotypes and, finally, their failure to complement the lethal alleles of the respective genetic loci. Our results show that although only a small fraction of the total transposant lines displayed patterned w expression, the genetic loci thus identified are those which play essential roles in pattern formation. Scopes of screens for genetic loci based on w reporter gene expression in adult eyes are discussed.     

Complete DNA sequence of the mitochondrial genome ofCepaea nemoralis (Gastropoda: Pulmonata)

The nucleotide sequence of a mitochondrial genome of the pulmonate gastropod molluscCepaea nemoralis has been determined. Contained within the 14,100 basepairs (bp) are the two ribosomal RNA genes and 13 protein coding genes typical of metazoan mitochondrial genomes. TheCepaea mtDNA does contain a gene for ATPase subunit 8, like the clausiliid pulmonate,Albinaria, and the chiton,Katharina, but unlike the bivalve mollusc,Mytilus. The mitochondrial genetic code ofCepaea is proposed to be the same as that ofMytilus, Katharina, andDrosophila. Only 14 putative tRNA genes are presented, although there is sufficient unassigned sequence to encode the remainder of the expected total of 22 tRNA genes. These 14 tRNA genes are a mixture of standard cloverleaf structures and nonstandard structures containing TV replacement loops as seen in nematode and mosquito mitochondrial genomes. If the eight unidentified tRNA genes are indeed present, very little unassigned sequence would remain to serve as a control region. Genes are transcribed from both strands of the molecule. Base composition is the least biased for any reported animal mitochondrial genome and is also very little skewed between strands using measures independent of base composition. TheCepaea mitochondrial gene order is quite unlike that of any other reported metazoan mtDNA, with the exception of the recently reported partial sequences ofAlbinaria. No gene bound-aries are shared among all the reported molluscan taxa, demonstrating a complete lack of conservation of mitochondrial gene order across the phylum Mollusca.     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.     

Single gene mutations in Drosophila: what can they tell us about the evolution of sexual behaviour?

The molecular analysis of specific mutant genes that affect the courtship behaviours of Drosophila melanogaster males and females is discussed in the light of the possibility that they may contribute to mate choice. There is clear evidence that some genes can act as a reservoir of species-specific behaviour, particularly for the male actions during courtship. However, to date there has not been a single genetic locus that has been isolated at the molecular level and shown to be associated with a change in female preference. There are some promising avenues of exploration, in that recent genetic analyses suggest that a small number of genes may make major contributions to female preferences. Finally a candidate gene approach is advocated in which orthologous genes from other species of Drosophila are used as natural mutations, and transformed into D. melanogaster hosts to investigate whether they carry species-specific mating information of the donor.     

Drosophila acidic ribosomal protein rpA2: Sequence and characterization

A cDNA encoding the Drosophila melanogaster acidic ribosomal protein rpA2 was cloned and sequenced. rpA2 is homologous to the Artemia salina acidic ribosomal protein eL12′. In situ hybridization to salivary gland polytene chromosomes localizes the rpA2 gene to band 21C. It is a single copy gene, with an mRNA of 0.8 kb. Two-dimensional gel electrophoresis of Drosophila ribosomal proteins followed by immuno-blotting showed that the rpA2 protein has an apparent relative mobility in SDS of 17 kD and an isoelectric point less than pH 5.0. Although the Drosophila gene rp21C may be the same as rpA2, the reported sequences differ. Comparisons of the aligned nucleotide sequences coding for the acidic ribosomal proteins rpA1 and rpA2 of Drosophila with those of other eukaryotes support the view of two separate, though closely related, groups of acidic proteins. Comparison with the Artemia homologues suggests that nucleotide identity may have been conserved by some constraint that acts in addition to the requirement for substantial similarity of amino acid sequences. © 1993 Wiley-Liss, Inc.     

SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing

This review summarizes genetic, molecular and biochemical studies of the SU(VAR)3-9 protein and the evidence for its key role in heterochromatin formation and heterochromatic gene silencing. The Su(var)3-9 locus was first identified as a dominant modifier of position-effect variegation (PEV) in Drosophila melanogaster. Together with Su(var)2-5 and Su(var)3-7, Su(var)3-9 belongs to the group of haplo-suppressor loci which show a triplo-dependent enhancer effect. All three genes encode heterochromatin-associated proteins. Su(var)3-9 is epistatic to the PEV modifier effects of Su(var)2-5 and Su(var)3-7, and it also dominates the effect of the Y chromosome on PEV. These genetic data support a central role of the SU(VAR)3-9 protein in heterochromatic gene silencing, one that is correlated with its activity as a histone H3-K9 methyltransferase (HMTase). In fact, SU(VAR)3-9 is the main chromocenter-specific HMTase of Drosophila. SU(VAR)3-9 and HP1, the product of Su(var)2-5, are main constituents of heterochromatin protein complexes and the interaction between these two proteins is interdependent. Functional analysis in fission yeast, Drosophila and mammals demonstrate that SU(VAR)3-9-dependent gene silencing processes are conserved in these organisms. This is also demonstrated by the rescue of Drosophila Su(var)3-9 mutant phenotypes with human SUV39H1 transgenes.     

Nucleotide divergence of the rp49 gene region between Drosophila melanogaster and two species of the Obscura group of Drosophila

Summary A 2.1-kb SStI fragment including the rp49 gene and the 3 end of the -serendipity gene has been cloned and sequenced in Drosophila pseudoobscura. rp49 maps at region 62 on the tip of chromosome II of this species. Both the coding and flanking regions have been aligned and compared with those of D. subobscura. There is no evidence for heterogeneity in the rate of silent substitution between the rp49 coding region and the rate of substitutions in flanking regions, the overall silent divergence per site being 0.19. Noncoding regions also differ between both species by different insertions/deletions, some of which are related to repeated sequences. The rp49 region of D. pseudoobscura shows a strong codon bias similar to those of D. subobscura and D. melanogaster. Comparison of the rates of silent (K _S ) and nonsilent (K _a ) substitutions of the rp49 gene and other genes completely sequenced in D. pseudoobscura and D. melanogaster confirms previous results indicating that rp49 is evolving slowly both at silent and nonsilent sites. According to the data for the rp49 region, D. pseudoobscura and D. subobscura lineages would have diverged some 9 Myr ago, if one assumes a divergence time of 30 Myr for the melanogaster and obscura groups.Offprint requests to: C. Segarra     

The determination of sense organs in Drosophila: interaction of scute with daughterless

Summary Several genetic loci have been implicated in the formation of the peripheral nervous system during Drosophila embryogenesis. As a first step towards understanding the functional interrelationships between these genes, we have searched for dominant interactions between deficiencies for the achaete-scute complex (AS-C), daughterless (da) and six other regions necessary for peripheral neurogenesis in the embryo. We have found that adult flies doubly heterozygous for deletions of AS-C and of da, or of AS-C and a small region on the fourth chromosome, exhibit characteristic bristle defects, suggesting that these genes cooperate to form sense organs both in the embryo and in the adult.     

Genetic control of translational fidelity in yeast: molecular cloning and analysis of the allosuppressor gene SAL3

Summary The fidelity of translation in the yeast Saccharomyces cerevisiae is controlled by a number of gene products. We have begun a molecular analysis of such genes and here describe the cloning and analysis of one of these genes, SAL3. Mutations at this locus, and at least four other unlinked loci (designated SAL1-SAL5), increase the efficiency of the tRNA ochre suppressor SUQ5, and are thus termed allosuppressors. We have cloned the SAL3 gene from a yeast genomic library by complementation of a sal3 mutation. Integration of the cloned sequence into the yeast chromosome was used to confirm that the SAL3 gene had been cloned. SAL3 gene is present in a single copy in the yeast genome, is transcribed into a 2.3-kb polyadenylated mRNA and encodes a protein of M_r 80 000. The size of the SAL3 gene product strongly suggests that it is not a ribosomal protein.     

The suppressor of forked locus in Drosophila melanogaster: genetic and molecular analyses

The suppressor of forked, su(f) locus is one of a class of loci in Drosophila whose mutant alleles are trans-acting allele-specific modifiers of transposable element-insertion mutations at other loci. Mutations of su(f) suppress gypsy insert alleles of forked and enhance the copia insert allele white apricot. Our investigations of su(f) include genetic and molecular analyses of 19 alleles to determine the numbers and types of genetic functions present at the locus. Our results suggest the su(f) locus contains multiple genetic functions. There are two distinct modifier functions and two vital functions. One modifier function is specific for enhancement and the other for suppression. One vital function is required for normal ecdysterone production in the third larval instar, the other is not. We present a restriction map of the su(f) genomic region and the results of an RFLP analysis of several su(f) alleles.     

Elucidation of origin of the present day hybrid banana cultivars using the 5′ETS rDNA sequence information

In the present study, our intention was to elucidate the genetic relation of M. acuminata subspecies and analyse the diversity of the M. balbisiana gene-pool using nuclear ribosomal gene loci based marker system. Additionally the obtained information allowed elucidating the structure and ancestry of the nuclear genomes of diploid and triploid cultivars. By establishing the nucleotide sequence of the rDNA locus for M. acuminata and partially for M. balbisiana and their comparative analysis revealed that the 5′ETS region was the most divergent between acuminata and balbisiana genomes. Based on the SNP sites identified in this region a PCR based system was built, which revealed four gene-pools among M. acuminata wild types, while M. balbisiana showed no sequence divergence. The developed markers proved to be a powerful tool in the identification of the acuminata component of diploid and triploid hybrid cultivars and discovery of unexpected genotypes.     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam