Strain typing with ISLpl1 in lactobacilli

Twenty-seven Lactobacillus plantarum ssp. plantarum, 11 Lactobacillus paraplantarum and five Lactobacillus casei-related strains, isolated from various autochthonous Serbian and Montenegro-fermented foods, were identified using phenotypical characterization and current PCR methods based on PCR of the recA gene or the 23S-5S rRNA gene intragenic spacer (IS) region. The strains were genotypically characterized by a new method based on the insertion sequence element ISLpl11 that grouped these lactobacilli into 10 IS-fingerprinting groups. Between six and 23 copies of the ISLpl1 were found in each strain and the ISLpl1-fingerprint groups correlated well with the origin of the strains. The method proved suitable for strain typing of lactic acid bacteria at the infraspecies level.     

Random Genome Sequencing of Ralstonia solanacearum Strain IVIA 1602 and Comparative Analysis with Strain GMI1000

Ralstonia solanacearum is a β‐proteobacterium which affects several hundred plant species and provokes important agronomic losses. Five biovars of this bacterium have been described and they show behavioural differences. In this study a random sequencing of the genome of R. solanacearum strain IVIA 1602 (race 3, biovar 2), isolated from potato, was performed. The resulting 730 Genomic Survey Sequences (GSSs), representing 6.38% of the complete genome, were compared against the completely sequenced genome of strain GMI1000 (race 1, biovar 3), isolated from tomato, which is the only strain of this species sequenced until now. This comparative analysis showed, as expected, a high degree of similarity, but it also revealed strain‐specific regions of the genome as well as a number of insertion/deletion events and chromosomal rearrangements. All together, this comparative analysis gives an overview of the genomic divergence between these two biovars of the R. solanacearum species complex.     

Strain of Yoshida sarcoma with induced pafencil resistance

Pafencyl, a new antineoplastic drug, approved for use in the USSR, was administered orally to Wistar rats bearing subcutaneous Ioshida's sarcomas (during 5 days, 1 to 10 mg/kg bw). Administration of the drug during several days induced drug resistance. Since the 28th transplant generation accompanied by pafencyl administration drug resistance became stable and the maximal tolerable dose (MTD) 40 mg/kg, which causes regression of the sensitive strain, was ineffective. In rats bearing bilaterally transplanted resistant and sensitive tumors, pafencyl in MTD and lower doses completely inhibited the growth of the sensitive line with a zero or stimulation effect on the tumor-resistant one. The resistance index was 30.9. The drug resistance remained unchanged for 2 years under cryopreservation in liquid nitrogen at -196 degrees C. This tumor-resistant model can be used for studying the mechanisms of drug resistance, cross resistance and preclinical testing of new drugs.     

紫外线诱变选育高产PHB解聚酶的菌株

以降解聚-β羟基丁酸酯(PHB)的青霉(Penieillium sp．)DS9713a为出发菌株,通过紫外线(UV)诱变分生孢子,采用透明圈初筛和摇瓶复筛,获得酶活高于原始菌株的突变株5株,其中DS9713a-CS01突变株的PHB解聚酶活力高于对照97．42％,并对其酶学性质进行了初步研究。     

Genome Comparison of a Nonpathogenic Myxoma Virus Field Strain with Its Ancestor,the Virulent Lausanne Strain

One of the best-studied examples of host-virus coevolution is the release of myxoma virus (MV) for biological control of European rabbits in Australia and Europe. To investigate the genetic basis of MV adaptation to its new host, we sequenced the genome of 6918, an attenuated Spanish field strain, and compared it with that of Lausanne, the strain originally released in Europe in 1952. Although isolated 43 years apart, the genomes were highly conserved (99.95% identical). Only 32 of the 159 MV predicted proteins revealed amino acid changes. Four genes (M009L, M036L, M135R, and M148R) in 6918 were disrupted by frameshift mutations.Myxoma virus (MV), the causative agent of myxomatosis, belongs to the Leporipoxvirus genus of the Poxviridae family (9). Two distinct types of MV have been identified: South American MV, which circulates in Sylvilagus brasiliensis, and Californian MV, which circulates in Sylvilagus bachmani. Each virus is highly adapted to its host, causing a benign cutaneous fibroma at the site of inoculation. Both types of MV infect the European rabbit (Oryctolagus cuniculus), causing myxomatosis. The Californian strain MSW is more virulent for European rabbits than South American strains such as SLS or Lausanne (54). Another leporipoxvirus, Shope fibroma virus (SFV), is found in eastern North America in Sylvilagus floridanus. SFV protects European rabbits against myxomatosis (24), and it is routinely used as a vaccine.One of the best-studied examples of host-virus coevolution is the use of MV for biological control of European rabbits (22, 23, 29). It is particularly unusual because the precise time the virus was released is known, and the original viruses are available for comparison with current strains. MV (the SLS strain) was deliberately released in Australia in 1950 and soon after (1952) in France (the Lausanne strain), whence it rapidly spread across Europe, and it has become endemic since then. For almost 60 years, a complex coevolution of host and virus has occurred, characterized by the emergence of attenuated viral strains and rabbits selected for resistance to MV (11, 12, 30).The MV Lausanne strain and SFV have been completely sequenced (13, 61). MV encodes 171 genes, versus 165 encoded by SFV. The genetic information is highly conserved between the two viruses. Recently, preliminary sequencing of the MSW strain indicated that the major genomic differences with the Lausanne strain localize at the left terminal end of the MSW genome (31). In MSW, the terminal inverted repeats (TIRs) are extended, causing the duplication of five complete open reading frames (ORFs), which are present as a single copy near the right TIR in the Lausanne strain (9). To date, little molecular analysis concerning the adaptation of MV to its new host has been performed. Studies involving Australian field strains found small differences with reference to the SLS and Lausanne strains (49, 50), suggesting that adaptation (and the concomitant attenuation) of MV is not associated with major genetic changes such as large deletions. This finding is in contrast to what has been reported for attenuated poxviruses obtained by extensive cell culture passaging, which usually present substantial genomic deletions or rearrangements (5, 25, 36, 47, 48).Strain 6918 is a naturally attenuated MV isolated in Spain in 1995 (7). It is therefore a descendant of the Lausanne strain recovered after 43 years of continuous evolution in the field. It has been used for the development of a “transmissible vaccine” intended to protect wild-rabbit populations against both myxomatosis and rabbit hemorrhagic disease virus (RHDV) in Spain, where the European rabbit plays a key role in the Mediterranean ecosystems (18). For this purpose, a recombinant virus, 6918VP60-T2, was constructed by inserting the capsid gene of RHDV into the genome of strain 6918 (4, 6, 7, 56, 57). The genomes of 6918 and 6918VP60-T2 have been sequenced. Here we report the results of our comparison of the genomic sequences of Lausanne and 6918. To our knowledge, this is the first comparative genomic analysis involving two poxvirus field strains linked by a clearly recorded lineage, one being fully virulent and the other virtually nonpathogenic. The results provide relevant insights into the mechanisms of MV attenuation that occurred as a consequence of the adaptation of the virus to its new host.     

柔嫩艾美耳球虫地克株利抗药株与敏感株孢子化卵囊的蛋白质差异分析

利用双向电泳技术,对本实验室诱导保存的柔嫩艾美耳球虫地克株利抗药株与敏感株的蛋白质表达图谱进行差异比较和分析,发现两者之间差异有5个蛋白质斑点,利用MALDI_TOF_TOF质谱技术对其中4个差异明显的蛋白质斑点进行分析鉴定,获得4个明确的肽质量指纹图谱,通过在NCBInr数据库中检索分析,确定了其中2个蛋白质分别为球虫子孢子表面抗原TA4和热休克蛋白Hsp70 ,另外两种为真核细胞的功能蛋白。上述蛋白的鉴定将对球虫的抗药性产生机理和柔嫩艾美耳球虫地克株利抗药株的分子标志物提供了研究方向。     

曲酸高产菌株的诱变选育

以紫外线和氮离子注入作为诱变手段对曲酸产生菌黄曲霉CICC2242进行诱变处理,筛选出一株高产菌株N83。曲酸产量由初始的12．4g／L提高到19．4g／L,提高了56．5％;遗传稳定性实验结果表明,N83遗传性状稳定良好。该结果表明氮离子注入诱变是获得高产曲酸的有效途径。     

Plant Transformation by Coinoculation with a Disarmed Agrobacterium tumefaciens Strain and an Escherichia coli Strain Carrying Mobilizable Transgenes

Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.     

絮凝性强的优良面包酵母菌株的选育

通过初筛、单倍体分离、DES诱变、絮凝基因的克隆表达及杂交等育种技术成功构建了高生物量、耐高糖、强絮凝的优良面包酵母菌株(Saccharomyces cerevisiae) ZLTH58(MATa/α,leu,FLO1)。菌株ZLTH58具有双亲的优良性状,遗传性状稳定。对其生物量、耐高糖能力、絮凝特性进行了检测,结果表明,菌株ZLTH58的生物量是原始亲株BL56的1.21倍;耐高糖能力优于原始亲株BL61;絮凝性能明显优于原始亲株BL56和BL61。对其培养条件进行了优化,在优化的培养条件下,生物量可以达到83.06g/L,为初始培养条件下的1.35倍。