Comparative study on the use of specific and heterologous microsatellite primers in the stingless bees Melipona rufiventris and M. mondury (Hymenoptera, Apidae)

Due to their high degree of polymorphism, microsatellites are considered useful tools for studying population genetics. Nevertheless, studies of genetic diversity in stingless bees by means of these primers have revealed a low level of polymorphism, possibly the consequence of the heterologous primers used, since in most cases these were not specifically designed for the species under consideration. Herein we compared the number of polymorphic loci and alleles per locus, as well as observed heterozygosity in Melipona rufiventris and M. mondury populations, using specific and heterologous primers. The use of specific primers placed in evidence the greater frequency of polymorphic loci and alleles per locus, besides an expressive increase in observed heterozygosity in M. rufiventris and M. mondury, thereby reinforcing the idea that populational studies should be undertaken by preferably using species-specific microsatellite primers.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.     

Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was detected within the breeds. Nucleotide substitution was determined in the HindIII polymorphic site by DNA cloning and sequencing; this is C----T at position 10126 of the URF-3 region. Furthermore, the MspI and TaqI polymorphic sites were located on the physical map.     

应用CRISPR/Cas13b编辑技术治疗TNNT2R141W转基因小鼠的扩张型心肌病

本研究旨在应用CRISPR/Cas13b系统对TNNT2R141W转基因扩张型心肌病（dilated cardiomyopathy,DCM）小鼠（DCM小鼠）进行探索性治疗,尝试发现治疗扩张型心肌病的一种新方式,为CRISPR/Cas13b系统在体内应用提供实验基础。随机设计11种Cas13b-TNNT2 gRNA并成功构建表达质粒,把它和人源TNNT2过表达质粒共同转染到293T细胞中,通过实时定量PCR（quantitative real-time PCR,Q-PCR）检测人源TNNT2 mRNA的表达水平。结果显示,gRNA 2引导Cas13b敲低目标基因的效率最高,达到80%（P<0.0001）。把gRNA2表达质粒包装到慢病毒载体中转导出生后1天的DCM小鼠原代心肌细胞,Q-PCR检测结果表明CRISPR/Cas13b系统对人源TNNT2 mRNA的敲低效率达到55%（P<0.01）。把PspCas13b和gRNA2的表达载体分别包装到AAV9病毒载体中,然后将200 μL 约1×1012 AAV9病毒颗粒通过尾静脉注射到4月龄DCM小鼠体内,待注射小鼠发育至5月龄时,Q-PCR检测结果显示,AAV9+DCM组TNNT2R141W表达水平较未注射组对照明显下降至40%（P<0.01）。对5月龄野生型（WT）、DCM（未注射病毒组）和AAV9+DCM（基因组编辑工具注射组）三组小鼠的心脏形态、心功能、心肌纤维化和心力衰竭等表型的观察结合显示：DCM小鼠的心脏形态异常,而AAV9+DCM小鼠心脏形态趋于正常;对三组小鼠的心脏进行超声心动图并对心功能指标进行统计发现,DCM组较WT组小鼠的左心室射血分数（left ventricular percent ejection fraction,LV EF%）、左心室短轴缩短率（left ventricular percent fractional shortening,LV FS%）分别下降了50.4%（P<0.0001）,55.1%（P<0.0001）,而AAV9+DCM组较DCM组小鼠的LV EF%、LV FS%分别上升了66.5%（P<0.01）,77.0%（P<0.01）;通过Q-PCR和天狼星红染色检测三组小鼠的心脏纤维化程度,结果显示DCM组较WT组小鼠的Col3a1和Postn两种纤维化基因,分别高表达5.2倍（P<0.001）、4.5倍（P<0.01）,而AAV9+DCM组较DCM组小鼠两种基因表达分别下降了2.0倍（P<0.05）、1.4倍（NS）,天狼星红染色结果显示纤维化区域明显下降;通过Q-PCR和蛋白质免疫印迹分别检测三组小鼠的心脏心力衰竭基因Nppb mRNA和Nppa蛋白质的表达水平,结果表明DCM组较WT组小鼠Nppb mRNA表达上升14.2倍（P<0.01）,而AAV9+DCM组较DCM组小鼠Nppb mRNA表达明显下降下降2.8倍（P<0.05）,Nppa蛋白质表达趋势与Nppb相同。把gRNA 5和含有R141W突变（gRNA 5T）和正常的TNNT2 mRNA（gRNA 5V）序列分别组合转染到293T细胞中,通过Q-PCR检测两种序列mRNA的表达水平。结果显示,gRNA 5T序列表达效率为30%（P<0.0001）,而并未检测到gRNA 5V mRNA的敲低。本研究通过设计靶向TNNT2R141W mRNA的gRNA,特异性敲低TNNT2R141W转基因小鼠体内突变的mRNA,有效改善了转基因小鼠的心功能,为临床进一步探索扩张型心肌病的治疗奠定了实验室基础。     

DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera,Meliponini)

We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.     

Mitochondrial DNA polymorphism in Russians from west Siberia.

The mitochondrial DNA (mtDNA) of 60 Russians from West Siberia was analyzed with the following restriction enzymes: BamHI, HindIII, PstI, PvuII and SacI that recognize 6 bp. The observed restriction fragment length polymorphisms (morphs) were classified into 13 types of distinct cleavage patterns (mitotypes). The distributions of the mtDNA morphs were compared with those characteristic of some other human populations.     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.     

Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.     

Restriction endonuclease analysis of mitochondrial DNA of three farm animal species: Cattle, sheep and goat

Five restriction endonucleases (HindIII, BgIII, EcoRI, EcoRV and BamHI) were employed to analyse mitochondrial DNA of cattle, sheep and goat. The results showed completely different restriction patterns of mtDNA among the three bovid species. A total of 11, 16, and 17 restriction fragments in cattle, sheep and goat respectively, were detected by the five restriction endonucleases. Average total sizes of mtDNA of cattle, sheep and goat were found to be 16.49 ± 0.18, 16.30 ± 0.25 and 16.44 ± 0.08 kb, respectively. The mtDNA cleavage patterns were identical for all seven individuals belonging to two cattle breeds and for 10 individuals from one sheep breed.     

DNA methylation in stingless bees with low and high heterochromatin contents as assessed by restriction enzyme digestion and image analysis.

BACKGROUND: The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen-DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence -CCGG-, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated. METHODS: Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to the Feulgen reaction, and analyzed by automatic scanning microspectrophotometry. RESULTS: The Feulgen-DNA values for the heterochromatin of M. rufiventris and for the small heterochromatin and some euchromatin domains of M. quadrifasciata mostly decreased after treatment with Msp I, but were unchanged after treatment with Hpa II. CONCLUSION: CpG methylation, although detected in diverse chromatin compartments in different bee species, may induce silencing effects required for the same cell physiology.     

Mitochondrial and ribosomal DNA variation among members of the Anopheles quadrimaculatus (Diptera: Culicidae) species complex.

The extent of intra- and inter-specific variation in mitochondrial DNA and nuclear ribosomal RNA gene restriction sites was determined for the four sibling species of the Anopheles quadrimaculatus complex. Individual mosquitoes were identified by allozyme analysis according to previously published keys, and the total genomic DNA of these same individuals was then cleaved with restriction enzymes. Restriction maps of mitochondrial DNA, including the positions of variable sites, were constructed for each species. No evidence for interspecific hybridization was found in the populations surveyed. There was little variation in restriction patterns within any given species, but differences occurred among the four. Three restriction enzymes (AvaI, HindIII, and PvuII) yielded species-specific DNA restriction patterns for the mitochondrial DNA, while AvaI and HindIII produced diagnostic patterns for the ribosomal DNA. Thus, restriction patterns were very useful for detecting cryptic species but less appropriate than isozymes for studying genetic structure of populations within species.     

猕猴属五个种mtDNA多态性研究

本文以10种限制性内切酶研究猕猴属5个种(Macaca mulatta.M.nemestrina.M.assemensis.M.thibetana,M arctoides)线粒体DNA进化。在13个个体中,共检出8种限制性类型。恒河猴种内存在广泛的线粒休DNA限制性片段长度多态性(RFLP)。结合日本猴(M.fuscata)的有关资料,构建了猕猴属6个种的分子系统树,并给出各个种的分化时间。结果表明,这6个种可分成4个类群,熊猴和藏酋猴、恒河猴和日本猴之间的遗传距离较近,可分别划为同一类群,红面猴与其他5种猴的遗传距离最远,在系统发生上分离最早。     

Restriction endonuclease analysis of total deoxyribonucleic acid of Mycobacterium tuberculosis H37RV (ATCC 27294) and of M. bovis (ATCC 19210)

Total DNA from two slowly-growing pathogenic mycobacterial species propagated in vitro was isolated, digested with each of 34 restriction endonucleases and analyzed by agarose gel electrophoresis. The most distinct profiles for M. tuberculosis (ATCC 27294) and for M. bovis (ATCC 19210) were obtained respectively using (BamHI, DraI, ClaI, EcoRI, EcoRV, HindIII, HpaI, SalI, SmaI, XbaI, and XmaI). The patterns produced for these strains were reproducible and distinguishable from each other. However, with several enzymes the patterns for M. tuberculosis and M. bovis were similar. Evidence was obtained for the presence of dam and dcmI methylations in the DNA of each mycobacterial species.     

Comparison of restriction fragment length polymorphisms of ribosomal DNA between Diphyllobothrium nihonkaiense and D. latum.

Restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA) were compared between Diphyllobothrium latum and D. nihonkaiense using seven kinds of restriction endonucleases. No intra-specific variation in restriction fragment profiles was shown within both species of Diphyllobothrium. Digestion of the genomic DNA with three endonucleases. SmaI, HinfI and HhaI, provided one or two different bands between two species, although the hybridization patterns generated with the others. HindIII, XbaI, StyI and HaeIII, were the same in both. RFLPs in the digested profiles with SmaI, HinfI and HhaI could be used as species-specific markers even if only fragments of strobilae with morphological similarity were available. Other cestodes, Spirometra erinacei and Taenia saginata, used as controls showed quite different restriction fragment patterns with all the enzymes used.     

Nucleotide sequence variants of Rattus norvegicus mitochondrial DNA

The mitochondrial DNA (mtDNA) molecules of different albino, domesticated rats (Rattus norvegicus) of the SASCO colony are of two kinds (SASCO-1 and SASCO-2) in regard to their sensitivity at certain sites to a number of restriction enzymes. MtDNA molecules from Utah wild R. norvegicus (Wild-UT) have sensitivities to restriction enzymes which differ at some sites from either SASCO-1 or SASCO-2 mtDNA molecules. Four single nucleotide differences were found among the HindIII F fragments (169 nucleotides) of SASCO-1, SASCO-2, and Wild-UT mtDNAs. Arguments are presented in favor of the interpretation that each variant nucleotide is the third nucleotide of the codon containing it, and that none of the four differences would result in a difference in the respective amino acid translated.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Comparative restriction endonuclease analysis and molecular cloning of plastid DNAs from wild species and cultivated varieties of the genus Beta (L.)

Summary A phyletic tree of the genus Beta has been constructed based on EcoRI and PstI plastid DNA restriction patterns of eight species from three sections of the genus. In contrast to the remarkable morphological variability of the varieties of B. vulgaris the restriction patterns of the plastid DNA of this species were found to be almost identical. The comparison of plastic DNAs of B. vulgaris crassa fertile and sterile lines with 13 different restriction enzymes revealed only a single fragment polymorphism in the HindIII patterns. Hybridization analyses in the plastidal rDNA region revealed an interesting loss of an EcoRI restriction site in all cultivated B. vulgaris varieties in contrast to wild species. The results of the construction of clone banks for SalI and BamHI fragments of plastid DNA from fertile B. vulgaris crassa are reported and difficulties in the cloning of specific fragments are discussed.     

Characterization of Staphylococcus species by ribosomal RNA gene restriction patterns

The rRNA gene restriction pattern sof 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindIII and EcoRI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilis inserted in a plasmid vector, pBR322. Fourty-four distinct HindIII patterns and 44 distinct EcoRI patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed with some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcus xylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g.S.aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.     

Ribosomal DNA variation in the grasshopper, Dichroplus elongatus.

We report an RFLP analysis of ribosomal DNA variation in natural populations of the grasshopper, Dichroplus elongatus, previously analyzed for mitochondrial DNA variation. DNA samples were digested with five restriction enzymes, BamHI, EcoRI, HindIII, PstI, and XbaI. BamHI was the only enzyme that showed no variation. The remaining enzymes showed fragment size variation at both intra- and interpopulation levels. Stepwise regression analysis revealed that the average number of length variants per individual is significantly associated with altitude. Moreover, the same analysis indicated that the frequency of some restriction variants exhibits a significant regression on both geographic and climatic variables. The intra- and interpopulation variability of rDNA was analysed by Lynch's and Hedrick's similarity indices using presence or absence of a fragment and band intensities, respectively. The corresponding neighbour-joining (N-J) trees based on Lynch's and Hedrick's genetic distances resulted in similar topologies. However, these trees were not in agreement with the N-J dendrogram obtained from mtDNA data previously reported by Clemente et al. (2000). The disagreement between mtDNA and rDNA phenograms along with the observed correlation between rDNA variability and geographical and climatic variables suggest some form of selection, besides genetic drift and migration, is involved in the pattern of rDNA variation.     

Correlation between ribosomal DNA polymorphism and electrophoretic enzyme polymorphism in Yersinia

Ribosomal DNA (rDNA) polymorphism was compared with electrophoretic enzyme polymorphism for the intra- and interspecies differentiation of Yersinia enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. aldovae, Y. frederiksenii and Y. kristensenii. DNA from 90 strains previously classified into six zymotypes (Y. enterocolitica and Y. frederiksenii) and into distinct enzyme electrophoretic patterns (the four other species) was digested with EcoRI or HindIII and analysed by Southern blotting. The six species were clearly differentiated from each other. In Y. enterocolitica, the subclassification of biotype 1 into zymotypes 1A and 1B was also reflected in the rDNA and the four other bio-zymotypes gave four different classes of restriction pattern. In Y. frederiksenii, both EcoRI and HindIII gave five distinct riboclasses which correlated with the zymotypes. In the four other species, the phenotype polymorphism appeared to be better correlated with the restriction fragment length polymorphism data in some enzymes than others. The data demonstrate that the inter- and intraspecies classification by rDNA polymorphism using two restriction enzymes is similar to that based on electrophoretic enzyme polymorphism. The analysis could be refined for taxonomic and epidemiological purposes by using other restriction enzymes.     

Sequence and RFLP analysis of the ITS2 ribosomal DNA in two Neotropical social bees, <Emphasis>Melipona beecheii</Emphasis> and <Emphasis>Melipona yucatanica</Emphasis> (Apidae,Meliponini)

Two stingless bees species of the genus Melipona, M. beecheii and M. yucatanica, are the only ones reported for the Yucatan Peninsula. The natural distribution of M. beecheii ranges from southern Mexico to Costa Rica, that of M. yucatanica from south Mexico to Guatemala. Colonies of both species occur in a variety of habitats and show adaptations to local conditions denoting the occurrence of ecotypes. The ITS2 of ribosomal DNA has been characterized in both species and its utility to discriminate among colonies has been investigated through RFLP experiments. The ITS2 region is unusually long, 1788 bp in M. beecheii and 1845 bp in M. yucatanica (including the 3′ end of the 5.8S gene and partial 5′ of the 28S gene). Mean nucleotide divergence between both ITS2 sequences is 16% (excluding sites with insertions/deletions) and 20% when the insertions/deletions are taken into account. The G+C content in both sequences is close to 53%. The PCR-RFLP assay was performed with 12 restriction enzymes on colonies of M. beecheii from Mexico (Yucatan, Campeche and Chiapas) Costa Rica, El Salvador and Guatemala, and of M. yucatanica from Mexico (Yucatan) and Guatemala. The restriction patterns obtained allow to discriminating colonies of both species with different origins. Both kinds of data are thus useful for assessing intra and interspecific genetic variability and for developing appropriate conservation strategies for these species. Received 20 June 2007; revised 31 August 2007; accepted 12 September 2007.