Prediction and phylogenetic analysis of mammalian short interspersed elements (SINEs)

The presence of repetitive elements can create serious problems for sequence analysis, especially in the case of homology searches in nucleotide sequence databases. Repetitive elements should be treated carefully by using special programs and databases. In this paper, various aspects of SINE (short interspersed repetitive element) identification, analysis and evolution are discussed.     

The Au family, a novel short interspersed element (SINE) from Aegilops umbellulata

A novel plant short interspersed nuclear element (SINE) was identified in the second intron of the acetyl CoA carboxylase gene of Aegilops umbellulata which has been designated ”Au”, for the host species in which it was discovered. Au elements have a tRNA-related region, direct flanking repeats, and a short stretch of T at the 3′ end, which are features common to Au and previously characterized SINEs. Au elements are detected in the genomes of several monocots and dicots by DNA dot hybridization and are also found in the tobacco genome by database searching. Au elements are present at an especially high copy number (approximately 10⁴ copies per haploid genome) in wheat and Ae. umbellulata. This suggests a recent amplification of Au in the Triticum and Aegilops species. In situ hybridization revealed a dispersed distribution of Au elements on wheat chromosomes. Au elements were amplified by PCR from monocot and dicot species and the phylogenetic relationships among Au elements were inferred. This phylogenetic analysis suggests amplification of Au elements in a manner consistent with the retrotransposon model for SINE dispersion. The high copy number of Au elements and their dispersed distribution in wheat are desirable characteristics for a molecular marker system in this important species. Received: 15 April 2000 / Accepted: 24 August 2000     

蜂群中不同亚家庭的工蜂寿命分析

在蜂群中,蜂王与多个雄蜂自然交尾,形成不同亚家庭.为了研究不同亚家庭中工蜂寿命是否有差异,我们以西方蜜蜂Apis mellifera L.为实验材料,随机从一群自然群中取270只刚孵化的工蜂,单独饲养于有蜜粉脾小蜂箱内,每天将自然死亡个体取出标记.并利用3对微卫星进行个体基因型分析,通过Matesoft软件划分亚家庭,然后分析了各亚家庭工蜂的自然寿命及生存曲线.结果表明:实验蜂群由12个亚家庭组成,其中2个亚家庭工蜂寿命与其他亚家庭存在显著差异(P＜0.05).     

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small,intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.     

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small,intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.     

Heterologous Amplification of Microsatellite Loci from Mouse and Rat in Oryzomyine and Thomasomyine South American Rodents

Eleven heterologous primers were investigated in 119 individuals of 11 species of rodents of the Oryzomyine and Thomasomyine groups. The animals were collected at four sites of the Cerrado, a dryland biome located on the Brazilian Plateau, all of them being karyotyped and taxonomically allocated according to the karyotype. Four of these primers, R47, R65, R75 (from Rattus), and ATP (from Mus) cross-amplified in at least one of these taxa, giving products of seven, nine, one, and three bands, respectively. These values are of the same order as others obtained when heterologous primers were amplified in other orders of mammals. Of the 20 products amplified in these two rodent groups by these four primers, only 7 of the bands were seen in a heteromorphic state (one individual presenting two bands), in two species (Rhipidomys aff. leucodactylus and Oryzomys megacephalus). The others occurred as monomorphic bands.     

A new subfamily of the grasshopper (Orthoptera: Acridoidea: Gomphoceridae) from the Tibetan Plateau of China

Abstract This paper reports a new subfamily, a new genus and a new species, that is, Pacrinae subfam. nov., Pacris gen. nov and Pacris xizangensis sp. nov in Gomphoceridae. The new subfamily is allied to Orinhippinae of Gomphoceridae and it differs from the latter by wings and tympanum absent. The new genus is similar to Orinhippus Uvarov, 1921 but differs from the latter in: (i) foveolae absent; (ii) tegmina absent; (iii) tympanum absent; (iv) hind margin of pronotum with incised in the middle. Type specimens are deposited in the Museum of Hebei University, Baoding, China.     

Ribokinase family evolution and the role of conserved residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia coli

Phosphofructokinase-2 (Pfk-2) belongs to the ribokinase family and catalyzes the ATP-dependent phosphorylation of fructose-6-phosphate, showing allosteric inhibition by a second ATP molecule. Several structures have been deposited on the PDB for this family of enzymes. A structure-based multiple sequence alignment of a non-redundant set of these proteins was used to infer phylogenetic relationships between family members with different specificities and to dissect between globally conserved positions and those common to phosphosugar kinases. We propose that phosphosugar kinases appeared early in the evolution of the ribokinase family. Also, we identified two conserved sequence motifs: the TR motif, not described previously, present in phosphosugar kinases but not in other members of the ribokinase family, and the globally conserved GXGD motif. Site-directed mutagenesis of R90 and D256 present in these motifs, indicate that R90 participates in the binding of the phosphorylated substrate and that D256 is involved in the phosphoryl transfer mechanism.     

Relationships among several species of the subfamily Gymnelinae (Zoarcidae) inferred from mitochondrial DNA sequence analysis

Nucleotide sequences of the mtDNA COI and cytochrome b genes were determined in Magadanichthys skopetsi, a member of the new monotypic genus Magadanichthys, endemic to the northern coast of the Sea of Okhotsk. Comparison of this species with other representatives of the subfamily Gymnelinae (family Zoarcidae) revealed high genetic similarity of M. skopetsi to Hadropareia middendorffii and considerable differences between these species and Gymnelopsis ochotensis.     

Additional box B of RNA polymerase III promoter in SINE B1 can be functional

Many genes of small RNAs and short interspersed elements (SINEs) are transcribed by RNA polymerase III due to an internal promoter that is composed of two boxes (A and B) spaced by 30-45 bp. Rodent SINE B1 originated from 7SL RNA, and a 29-bp tandem duplication took place in B1 at an early stage of its evolution. As a result of this duplication, an additional box B (named B') located at a distance of 79-82 bp from box A arose in SINE B1. Here we have shown that despite the unusually large distance between boxes A and B', they can form an active promoter. In chinchillas, guinea pigs, and other rodents belonging to clade Ctenohystrica, structure of the B' box was well preserved and closely resembles the canonical B box. One may suggest therefore, that box B' can functionally replace box B in those copies of B1 where the latter has lost activity due to mutations.     

PGU1 gene natural deletion is responsible for the absence of endo-polygalacturonase activity in some wine strains of Saccharomyces cerevisiae

The PGU1 gene encodes an endo-polygalacturonase enzyme in Saccharomyces cerevisiae. The literature reports that most S. cerevisiae strains possess this gene, despite a wide range of enzyme activity levels. Nevertheless, a few wine strains lack the PGU1 gene. We investigated the PGU1 locus sequence in these strains. The results indicated that the gene had been replaced by a partial Ty mobile element, whereas the gene promoter was still at the expected location. As all the strains lacking the PGU1 gene experienced the same phenomenon, it was tempting to hypothesize a common phylogenetic origin. However, fingerprints only allowed grouping of a few of them within one cluster.     

Species- and strain-specific probes derived from repetitive DNA for distinguishing Entamoeba histolytica and Entamoeba dispar

Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable species that are found in the human gut. Of the two, E. histolytica is considered to be pathogenic while E. dispar is nonpathogenic. To generate molecular probes to detect and distinguish between the two species, we utilized repeat sequences present in Entamoeba genome. We have developed probes and primers from rDNA episomes, and unidentified Entamoeba EST1 repeat for this purpose, and used them for dot blot hybridization and PCR amplification. To investigate the possible existence of invasive and noninvasive strains of E. histolytica, the ability to differentiate individual isolates is necessary. For this purpose, we have utilized a modification of the AFLP procedure called 'Transposon display,' which generates and displays large number of genomic bands associated with a transposon. We have used the abundant retrotransposon, EhSINE1, for this purpose,and demonstrated its potential as a marker to study strain variation in E. histolytica. This technique could suitably be employed in carrying out significant molecular epidemiological studies and large-scale typing of this parasite.     

Release of a plasma membrane-bound triaminopeptidase activity from mammalian cells by thermolysin.

Aminopeptidase and other enzyme activities on cellular surface were determined in the presence and absence of endopeptidases. Gly-Pro-Leu-AP activity was specifically released into the medium by thermolysin treatment, while the other activities retained on the cellular surface were markedly decreased. A similar phenomenon was also observed in rat liver membrane, mouse FM3A, spleen lymphocyte and other cells. Structural rearrangement of some protein components in the cell plasma membrane was suggested.     

Acidic lipase Lip I.3 from a Pseudomonas fluorescens‐like strain displays unusual properties and shows activity on secondary alcohols

Aims

Identification, cloning, expression and characterization of a novel lipase – Lip I.3 – from strain Pseudomonas CR‐611.

Methods and Results

The corresponding gene was identified and isolated by PCR‐amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0‐1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C‐terminal type I secretion signal and several putative Ca²⁺ binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca²⁺ ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols.

Conclusions

A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts.

Significance and Impact of the Study

The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis.     

Kakaducaris glabra gen. nov., sp. nov., a new freshwater shrimp from the Kakadu National Park,Northern Territory,Australia, Crustacea: Decapoda: Palaemonidae with the designation of a new subfamily Kakaducaridinae

A new species of palaemonid shrimp from the Northern Territory Kakadu National Park is described and illustrated. Kakaducaris glabra, gen, nov., sp. nov., can not be satisfactorily referred to the described subfamilies of the Palaemonidae and a new subfamily Kakaducaridinae is designated (see addendum).