Satellite DNA in the elm leaf beetle,Xanthogaleruca luteola (Coleoptera,Chrysomelidae): characterization,interpopulation analysis,and chromosome location

In this paper the satellite DNA (stDNA) of the phytophagous beetle Xanthogaleruca luteola is analyzed. It is organized in a tandem repeat of 149-bp-long monomers, has an AT content of 59%, and presents inverted internal repeats. Restriction analysis of the total DNA with methylation-sensitive enzymes suggests that this repetitive DNA is not methylated. Analysis of the electrophoretic mobility of stDNA on non-denaturing polyacrylamide gels showed that this stDNA is not curved. In situ hybridization with a biotinylated probe of the stDNA revealed a pericentromeric localization of these sequences in the majority of the meiotic bivalents. We have studied the stDNA of X. luteola from two populations with very distinct geographical origins. The sequence and phylogenetic analysis of monomers from these two populations showed that the repetitive element is conserved within the species. Putative gene conversion tracts are identified when the different monomers of the same population are compared. These results could indicate the existence of processes of homogenization that would extend these mutations to all the satellite repeats.     

Characterization of two divergently transcribed Dictyostelium gene pairs and identification of G-rich sequence element lying between them with the characteristics of a basal promoter element

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode coordinately expressed mRNA sequences that are inducible by extracellular cAMP. Both genes form part of divergently transcribed gene pairs. The gene proximal to CP1 is coordinately regulated and encodes a protein containing several potential zinc binding domains of the kind found in DNA binding proteins. The gene proximal to CP2 is a constitutively transcribed gene of unknown function. There are multiple, short, G-rich sequence elements between both gene pairs, and deletion of the pair of elements 200 nucleotides upstream from the CP2 gene abolishes cAMP-inducibility. A synthetic oligonucleotide, containing two copies of the G-rich element from the CP1 gene, will reconstitute cAMP-inducibility in the deletion mutant of the CP2 gene. This shows that the elements in the two genes are functionally homologous. Efficient induction requires at least two copies of the CP1 element, but their relative orientation is unimportant. Two copies in an inverted orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP-inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.     

A point mutation,R59G,within the HMG-SRY box in a female 45,X/46,X,psu dic(Y)(pter→q11::q11→pter)

We report a molecular and cytogenetic investigation of a psu dic(Yp) chromosome identified in blood and ovarian tissue from a female with mosaic karyotype 45,X/46,X,+ psu dic(Yp). FISH analysis showed that the psu dic(Yp) has two copies of the short arm, two centromeres and two copies of the proximal long arm. PCR analysis also confirmed the presence of the SRY gene and the Y centromere, and also confirmed the deletion of the Y-heterochromatic region. Because of the possibility of a mutation, a fragment of 609 bp of the SRY gene was sequenced from independent PCR products. The analysis of the sequence indicated the presence of two different copies of the gene: one presented a point mutation, R59G, within the HMG-box; the other had a sequence identical to that already published. Both sequences were found at a proportion of 1:1. The absence of a 46,XY cell line suggests that the rearrangement took place during gametogenesis or during the first division after fertilization. Also, the existence of different sequences of the SRYgene in the same Y chromosome suggests that the formation of the dicentric took place prior to the mutation of the SRY gene. To our knowledge, this is the first time that a mutation has been described in codon 59 within the HMG- SRY box, and also the first case of a psu dic(Yp) chromosome that displays two different copies of the SRY gene.     

Insertion of nonhomologous DNA into the yeast genome mediated by homologous recombination with a cotransforming plasmid

Bacterial plasmids containing no detectable homology with yeast DNA sequences were inserted into the yeast genome by cotransforming with a plasmid containing a yeast gene. Analysis of the yeast transformants confirmed that recombination events occurred between the prokaryotic sequences shared by the two plasmids and between the yeast sequences common to the cotransforming plasmid and to the genome. Multiple copies of the two plasmids, in both tandem and interspersed arrays, are inserted by this method. Populations of cells grown from individual transformants are heterogenous for the number of integrated sequences. The number of integrated bacterial sequences is greatly reduced after 100 generations of growth in the populations that initially contained large numbers of sequences, while it is stable in those populations that initially contained either a single or a small number of copies.     

Mouse ferritin H sequences map to chromosomes 3, 6, and 19

Human and rodent genomes contain multiple copies of ferritin H and L subunit sequences, although it is not yet clear whether there is more than one expressed gene for either of these subunits. We have isolated a cDNA corresponding to mouse ferritin H subunit and observed that the mouse genome contains three to four H-related sequences. This cDNA was used to establish the genomic location of mouse ferritin H subunit genes by chromosomal in situ hybridization. Metaphase chromosomes of concanavalin A-stimulated lymphocytes from a WMP male mouse were examined by in situ hybridization with 3H-labeled cDNA and the chromosomes were identified by R banding (fluorochrome-photolysis-Giemsa method). The results indicate that mouse ferritin H-related sequences map at chromosomes 3, 6, and 19. Homology of synteny between human and mouse suggests that the sequence on mouse chromosome 19 corresponds to the structural H gene.     

High frequency targeting of genes to specific sites in the mammalian genome

We corrected a defective gene residing in the chromosome of a mammalian cell by injecting into the nucleus copies of the same gene carrying a different mutation. We determined how the number, the arrangement, and the chromosomal position of the integrated gene, as well as the number of injected molecules influence the gene-targeting frequency. Recombination between the newly introduced DNA and its chromosomal homolog occurred at a frequency of 1 in 10(3) cells receiving DNA. Correction events were mediated by either double reciprocal recombination or gene conversion. This resulted in sequences in the genome being replaced by sequences of the introduced DNA or, in separate experiments, sequences in the incoming DNA being replaced by chromosomal sequences. Both point mutations and deletion mutations were corrected; however, the nature of the mutation carried by the respective sequence influenced whether the integrated or injected sequence was corrected.     

Multiple copies of a G-rich element upstream of a cAMP-inducible Dictyostelium gene are necessary but not sufficient for efficient gene expression.

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode co-ordinately expressed mRNA sequences which are inducible by extracellular cAMP. There are short, G-rich sequence elements upstream of both genes and we have previously shown that deletion of these elements from the CP2 gene abolishes cAMP-inducibility. We show here that the G-rich element from the CP1 gene is functionally homologous to that in the CP2 gene by reconstituting cAMP-inducibility in a deletion mutant of the CP2 gene using CP1-derived sequences. Both the CP1 and CP2 genes contain multiple G-rich elements. We show that efficient induction requires at least two copies of the CP1 element and that their relative orientation is unimportant. Two copies of an inverted relative orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP-inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.     

Adeno-associated virus general transduction vectors: analysis of proviral structures.

We used two kinds of adeno-associated virus (AAV) vectors to transduce the neomycin resistance gene into human cells. The first of these (dl52-91) retains the AAV rep genes; the second (dl3-94) retains only the AAV terminal repeats and the AAV polyadenylation signal (428 base pairs). Both vectors could be packaged into AAV virions and produced proviral structures that were essentially the same. Thus, the AAV sequences that are required in cis for packaging (pac), integration (int), rescue (res), and replication (ori) of viral DNA are located within a 284-base-pair sequence that includes the terminal repeat. Most of the G418r cell lines (73%) contained proviruses which could be rescued (Res+) when the cells were superinfected with the appropriate helper viruses. Some produced high yields of viral DNA; other rescued at a 50-fold lower level. Most of the lines that were Res+ (79%) contained a tandem repeat of the AAV genome (2 to 20 copies) which was integrated randomly with respect to cellular DNA. Junctions between two consecutive AAV copies in a tandem array contained either one or two copies of the AAV terminal palindrome. Junctions between AAV and cellular sequences occurred predominantly at or within the AAV terminal repeat, but in some cases at internal AAV sequences. Two lines were seen that contained free episomal copies of AAV DNA. Res+ clones contained deleted proviruses or tandem repeats of a deleted genome. Occasionally, flanking cellular DNA was also amplified. There was no superinfection inhibition of AAV DNA integration. Our results suggest that AAV sequences are amplified by DNA replication either before or after integration and that the mechanism of replication is different from the one used during AAV lytic infections. In addition, we have described a new AAV general transduction vector, dl3-94, which provides the maximum amount of room for insertion of foreign DNA and integrates at a high frequency (80%).     

Super-infections of Wolbachia in byturid beetles and evidence for genetic transfer between A and B super-groups of Wolbachia

Wolbachia are maternally inherited bacteria responsible for altering host reproduction. The two main groups found in insects, A and B, are based on molecular characterization using ribosomal, ftsZ, wsp (Wolbachia surface protein) or groE genes. We have used the wsp and ftsZ genes to study Wolbachia in byturid beetles. Byturus affinis contained a single copy of the ftsZ gene which grouped with A ftsZ sequences and a single copy of the wsp gene which grouped with B wsp sequences. This suggests that genetic exchange between A and B groups has occurred in the Wolbachia of this beetle. FtsZ and wsp sequences that were identical or nearly identical to those of B. affinis were found in B. tomentosus, suggesting that it also contains the same recombinant Wolbachia genotype. Most other byturids had more than one wsp sequence with at least one from the A and B groups, suggesting multiple copies of bacterial genes or multiple infections. B. ochraceus and B. unicolor both had four distinct wsp gene sequences. All the byturids had a closely related A wsp sequence and most a closely related B wsp sequence. Therefore, there appears to be an association between specific A and B wsp types.     

Vitellogenin of the parasitoid wasp, Encarsia formosa (Hymenoptera: Aphelinidae): gene organization and differential use by members of the genus

The vitellogenin (Vg) gene of the parasitoid wasp, Encarsia formosa (Hymenoptera: Aphelinidae), has been cloned and sequenced. The gene codes for a protein consisting of 1814 amino acids in seven exons. The position of the six introns in the E. formosa gene align with those inferred for the Vg gene of the honeybee, Apis mellifera. The position of two introns in the hymenopteran sequences are shared with every full-length insect Vg gene characterized to date. The deduced amino acid sequence of the E. formosa Vg gene most closely resembles that of the ichneumonid parasitoid, Pimpla nipponica (38% identity). The gene product, less the putative signal peptide, contains large quantities of serine (11.3% of total residues) but lacks the extensive polyserine tracts found in the Vgs of insects outside the apocritan Hymenoptera. The gene also codes for the highest level of lysine (9.5%), and lowest levels of phenylalanine (2.6%) and tyrosine (2.3%), observed in any insect Vg characterized to date. The mature gene product retains 12 cysteine residues in positions conserved in other insect Vgs. Ovary homogenates suggest that processed Vg is stored in the egg as an uncleaved molecule of approximately 200 kDa. Vg expression was examined in three additional Encarsia species. The protein was found in female E. sophia and E. luteola, but not in male E. luteola or female E. pergandiella. Despite extensive screening of a phage library prepared from E. pergandiella genomic DNA, a Vg gene was not detected in this species.     

A comparative study of convicilin storage protein gene sequences in species of the tribe Vicieae.

Convicilins, a set of seed storage proteins, differ from vicilins, a related group of seed storage proteins, mainly because of the presence of the N-terminal extension, an additional sequence of amino acids in the sequence corresponding to the first exon. Convicilins have been described only in species of the legume tribe Vicieae. One or two genes for convicilins have been identified in most species of this tribe. The genus Pisum is the main exception, since two genes have been identified in most of its species. Thirty-four new convicilin gene sequences from 29 different species (Lathyrus, Lens, Pisum, and Vicia spp.) have been analyzed here. Convicilin gene sequences are generally organized in 6 exons, but in some instances one of the internal introns (2nd or 4th) is lost. In these 29 species, the N-terminal extension is formed by a stretch of 99 to 196 amino acids particularly rich in polar and charged amino acids (on average, it contains 29.43% glutamic acid and 15.38% arginine residues). This N-terminal extension has the characteristics of an intrinsically unstructured region (IUR), one of the categories of protein "degenerate sequences". A comparative analysis indicates that the N-terminal extension sequence has evolved faster than the surrounding sequence, which is common to all vicilins, and it evolved mainly through a series of duplications of short internal sequences and triplet expansions, the predominant one being GAA. This agrees with the evolution of IURs, which is faster than the evolution of surrounding sequences and is mainly due to replication slippage and unequal crossover recombination. Alternative maximum-likelihood trees of phylogenetic relationships among the 29 Vicieae species based on the convicilin exon sequences are presented and discussed.     

Isolation and identification of Triticeae chromosome 1 receptor-like kinase genes (Lrk10) from diploid, tetraploid, and hexaploid species of the genus Avena.

The DNA sequence of an extracellular (EXC) domain of an oat (Avena sativa L.) receptor-like kinase (ALrk10) gene was amplified from 23 accessions of 15 Avena species (6 diploid, 6 tetraploid, and 3 hexaploid). Primers were designed from one partial oat ALrk10 clone that had been used to map the gene in hexaploid oat to linkage groups syntenic to Triticeae chromosome 1 and 3. Cluster (phylogenetic) analyses showed that all of the oat DNA sequences amplified with these primers are orthologous to the wheat and barley sequences that are located on chromosome 1 of the Triticeae species. Triticeae chromosome 3 Lrk10 sequences were not amplified using these primers. Cluster analyses provided evidence for multiple copies at a locus. The analysis divided the ALrk EXC sequences into two groups, one of which included AA and AABB genome species and the other CC, AACC, and CCCC genome species. Both groups of sequences were found in hexaploid AACCDD genome species, but not in all accessions. The C genome group was divided into 3 subgroups: (i) the CC diploids and the perennial autotetraploid, Avena macrostachya (this supports other evidence for the presence of the C in this autotetraploid species); (ii) a sequence from Avena maroccana and Avena murphyi and several sequences from different accessions of A. sativa; and (iii) A. murphyi and sequences from A. sativa and Avena sterilis. This suggests a possible polyphyletic origin for A. sativa from the AACC progenitor tetraploids or an origin from a progenitor of the AACC tetraploids. The sequences of the A genome group were not as clearly divided into subgroups. Although a group of sequences from the accession 'SunII' and a sequence from line Pg3, are clearly different from the others, the A genome diploid sequences were interspersed with tetraploid and hexaploid sequences.     

Colobus type C virus: molecular cloning of unintegrated viral DNA and characterization of the endogenous viral genomes of Colobus.

The unintegrated viral DNA intermediates of colobus type C virus (CPC-1) were isolated from infected human cells that were permissive for viral growth. There were two major species of DNA, linear molecules with two copies of the long terminal repeat and relaxed circles containing only a single long terminal repeat. In addition, there was a minor species (approximately 10%) composed of relaxed circles with two copies of the long terminal repeat. A restriction endonuclease map of the unintegrated DNA was constructed. The three EcoRI fragments of circular CPC-1 DNA were cloned in the EcoRI site of lambda gtWES . lambda B and then subcloned in the EcoRI site of pBR322. Using these subgenomic fragments as probes, we have characterized the endogenous viral sequences found in colobus cellular DNA. They are not organized in tandem arrays, as is the case in some other gene families. The majority of sequences detected in cellular DNA have the same map as the CPC-1 unintegrated DNA at 17 of 18 restriction endonuclease sites. There are, however, other sequences that are present in multiple copies and do not correspond to the CPC-1 map. They do not contain CPC-1 sequences either in an altered form or fused to common nonviral sequences. Instead, they appear to be derived from a distinct family of sequences that is substantially diverged from the CPC-1 family. This second family of sequences, CPC-2, is also different from the sequences related to baboon endogenous type C virus that forms a third family of virus-related sequences in the colobus genome.     

Multiple copies of coding as well as pseudogene c-mos sequence exist in three lacertid species

The analysis of a 581 bp section of the nuclear gene c-mos revealed multiple copies of putative functional sequences as well as pseudogenes in three closely related lacertid species Lacerta laevis, L. kulzeri and L. cyanisparsa. A phylogenetic analysis of c-mos in comparison with a molecular phylogeny based on the mitochondrial cytochrome b gene supports our findings. The study also provides new insights into the phylogenetic relationships of L. cyanisparsa and L. laevis.Pseudogenes of the three species share 11 single-nucleotide substitutions, a 1 bp deletion and a premature stop codon but differ by group-specific mutations. This result suggests that the c-mos gene has become duplicated and subsequently silenced already in the common ancestor of the three species. Sequence divergence suggests that the duplication and the loss of function occurred in the late Miocene/early Pliocene, i.e., about 5 million years ago. Indications of gene conversion are discussed.We suggest that future studies using c-mos for phylogenetic studies should provide evidence for the orthology of the sequences compared.     

Evidence that the N-terminal extension of the vicieae convicilin genes evolved by intragenic duplications and trinucleotide expansions.

This study was aimed to identify lentil (Lens culinaris subsp. culinaris) convicilin genes and to carry out a comparative analysis of these genes in the tribe Vicieae. Convicilins differ from vicilins, a related group of plant seed storage proteins, mainly by the presence of an additional sequence of amino acids in the sequence corresponding to the first exon, referred as the N-terminal extension. A single gene for convicilin, a component of legume seed storage proteins, was identified in the cultivated lentil. In this species, the N-terminal extension is formed by a stretch of 126 amino acids of which 59.2% are charged amino acids: 29.6% glutamic acid, 3.2% aspartic acid, 14.4% arginine, 8.8% lysine, and 3.2% histidine. This lentil convicilin sequence is similar to the sequence of convicilins in other species of the tribe Vicieae. However, the size of the N-terminal extension clearly differs among convicilins. Sequence comparison and phylogenetic analyses including convicilin and vicilin of Vicieae species indicated that the differentiation between vicilins and convicilins predated the differentiation of the two vicilin gene families (47- and 50-kDa vicilins), and that the N-terminal extension evolved mainly by a series of duplications of short internal sequences and triplet expansions, the predominant one being GAA.     

Repeated sequence sets in mitochondrial DNA molecules of root knot nematodes (Meloidogyne): nucleotide sequences, genome location and potential for host-race identification.

Within a 7 kb segment of the mtDNA molecule of the root knot nematode, Meloidogyne javanica, that lacks standard mitochondrial genes, are three sets of strictly tandemly arranged, direct repeat sequences: approximately 36 copies of a 102 ntp sequence that contains a TaqI site; 11 copies of a 63 ntp sequence, and 5 copies of an 8 ntp sequence. The 7 kb repeat-containing segment is bounded by putative tRNAasp and tRNAf-met genes and the arrangement of sequences within this segment is: the tRNAasp gene; a unique 1,528 ntp segment that contains two highly stable hairpin-forming sequences; the 102 ntp repeat set; the 8 ntp repeat set; a unique 1,068 ntp segment; the 63 ntp repeat set; and the tRNAf-met gene. The nucleotide sequences of the 102 ntp copies and the 63 ntp copies have been conserved among the species examined. Data from Southern hybridization experiments indicate that 102 ntp and 63 ntp repeats occur in the mtDNAs of three, two and two races of M.incognita, M.hapla and M.arenaria, respectively. Nucleotide sequences of the M.incognita Race-3 102 ntp repeat were found to be either identical or highly similar to those of the M.javanica 102 ntp repeat. Differences in migration distance and number of 102 ntp repeat-containing bands seen in Southern hybridization autoradiographs of restriction-digested mtDNAs of M.javanica and the different host races of M.incognita, M.hapla and M.arenaria are sufficient to distinguish the different host races of each species.     

The Agrocybe aegerita mitochondrial genome contains two inverted repeats of the nad4 gene arisen by duplication on both sides of a linear plasmid integration site.

The Agrocybe aegerita mitochondrial genome possesses two polB genes with linear plasmid origin. The cloning and sequencing of the regions flanking Aa-polB P1 revealed two large inverted repeats (higher than 2421 nt) separated by a single copy region of 5834 nt. Both repeats contain identical copies of the nad4 gene. The single copy region contains two disrupted genes with plasmid origin Aa-polB P1 and a small ORF homologous to a small gene described in two basidiomycete linear plasmids. The phylogenetic analyses argue in favor of a same plasmid origin for both genes but, surprisingly, these genes were separated by a mitochondrial tRNA-Met. Both strands of the complete region containing the two nad4 inverted copies and the tRNA-Met appear to be transcribed on large polycistronic mRNAs. A model summarizing the events that would have occurred is proposed: (1) capture of the tRNA by the plasmid before its integration in the mtDNA or acquisition of the tRNA gene by recombination after the plasmid integration, (2) integration of the plasmid in the mtDNA, accompanied by a large duplication containing the nad4 gene and (3) erosion of the plasmid sequences by large deletions and mutations.