The elimination of clover diseases by shoot tip culture

Shoot tip culture was used to eliminate white clover mosaic virus (WCMV) and red clover necrotic mosaic virus (RCNMV) from red clover, and clover phyllody disease (CP) and clover red leaf disease (CRL) from white clover. Shoot tips up to 2.4 mm (in some cases 3 mm) could regenerate plants free from the pathogens, but the efficiency of elimination, at least for WCMV and CRL, tended to decrease with increasing shoot tip size. The efficiency of plant regeneration from shoot tips generally improved with increasing tip size.     

Genetic control of retroviral disease in aging wild mice

Different populations of wild mice (Mus musculus domesticus) in Los Angeles and Ventura Counties were observed over their lifespan in captivity for expression of infectious murine leukemia virus (MuLV) and murine mammary tumor virus (MMTV) and for the occurrence of cancer and other diseases. In most populations of feral mice these indigenous retroviruses were infrequently expressed and cancer seldom occurred until later in life (>2 years old). MMTV was found in the milk of about 50% of wild mice, but was associated with only a low incidence (>1%) of breast cancer after one year of age. By contrast, in several populations, most notably at a squab farm near Lake Casitas (LC), infectious MuLV acquired at birth via milk was highly prevalent, and the infected mice were prone to leukemia and a lower motor neuron paralytic disease after one year of age. These two diseases were both caused by the same infectious (ecotropic)strain of MuLV and were the principal cause of premature death in these aging LC mice. A dominant gene called FV-4^R restricting the infection with ecotropic MuLV was found segregating in LC mice. Mice inheriting this FV-4^R allele were resistant to the ecotropic MuLV associated lymphoma and paralysis. The FV-4^R allele represents a defective endogenous MuLV provirus DNA segment that expresses an ecotropic MuLV envelope-related glycoprotein (gp70) on the cell surface. This FV-4^R encoded gp70 presumably occupies the receptor for ecotropic MuLV and blocks entry of the virus. The FV-4^R gene was probably acquired by the naturally occurring crossbreeding of LC feral mice with another species of feral mice (Mus castaneus) from Southeast Asia. The FV-4^R gp70 does not block entry of the amphotropic MuLV that uses a separate cell surface receptor. Therefore LC mice continued to be susceptible to the highly prevalent but weakly lymphogenic and nonparalytogenic amphotropic strain of MuLV. The study points out the potential of feral populations to reveal genes associated with specific disease resistance.     

Low temperature compartment formation in feline immunodeficiency virus-infected and uninfected feline kidney cells

Summary This study was to determine if feline immunodeficiency virus (FIV)-infected and uninfected Crandall feline kidney (CRFK) cells exhibited a low temperature (16°C) block in membrane trafficking between transitional endoplasmic reticulum and Golgi apparatus represented by intermediate compartment formation. Cells were cultured at different temperatures and membrane changes involving the Golgi apparatus and Golgi apparatus-associated membrane structures were monitored by electron microscopy and quantitated. With 30 min of incubation, membranes of the Golgi apparatus stack increased in amount at temperatures of 16°C and below compared to temperatures above 18°C. The increase was greatest along the major polarity axis as evidenced by an increased stack height. Neither the number of cisternae per stack nor the average stack diameter (width) was affected by temperature. The response was maximal between 15 and 30 min of low temperature treatment of the cells. Results with cells infected and uninfected with feline immunodeficiency virus were similar. The increase in stack height was due primarily to an increase of membranes at the cis face (cis Golgi apparatus network). At 18°C, membranes of the trans Golgi apparatus network accumulated suggesting that import from the cis Golgi network could proceed at this temperature, whereas exit from the trans Golgi network was still at least partially blocked. Also increased at 16°C and below were numbers of transition vesicles in the space between the Golgi apparatus and the transitional endoplasmic reticulum associated with the cis Golgi apparatus face. The results suggested interruption of the orderly flux of membranes into the Golgi apparatus at 16°C and below. Moreover, the block appeared to be reversible. Upon transfer from 16°C to 37°C, there was a time-dependent decrease in the accumulations of cis compartment membrane accompanied by a corresponding equivalent increase in the membranes of the trans Golgi apparatus compartment.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. II. Linkage relations and utility of a dominant gene for lethal systemic necrosis to soybean mosaic virus

Summary A single dominant factor, Hss, that conditions a rapid lethal necrotic response to soybean mosaic virus (SMV) has been identified in Phaseolus vulgaris L. cv. Black Turtle Soup, line BT-1. Inoculated plants carrying this factor developed pinpoint necrotic lesions on inoculated tissue followed by systemic vascular necrosis and plant death within about 7 days, regardless of ambient temperature. BT-1 also carries dominant resistance to potyviruses attributed to the tightly linked or identical factors, I, Bcm, Cam, and Hsw, so linkage with Hss was evaluated. No recombinants were identified among 381 F₃ families segregating for potyvirus susceptibility, thus if Hss is a distinct factor, it is tightly linked to I, Bcm, Cam, and Hsw. BT-1 was also crossed reciprocally with the line Great Northern 1140 (GN 1140) in which the dominant gene, Smv, for systemic resistance to SMV was first identified. Smv and Hss segregated independently and are co-dominant. The (GN 1140 x BT-1) F₁ populations showed a seasonal shift of the codominant phenotype. Evaluation of the (GN 1140 x BT-1) F₂ population under conditions where Smv is partially dominant allowed additional phenotypic classes to be distinguished. Pathotype specificity has not been demonstrated for either Smv or Hss. Genotypes that are homozygous for both dominant alleles are systemically resistant to the virus and in addition show undetectable local viral replication or and no seed transmission. This work demonstrates that a gene which conditions a systemic lethal response to a pathogen may be combined with additional gene(s) to create an improved resistant phenotype.     

Stable expression of antibiotic resistance genes using a promoter fragment of the U1 snRNA gene

As U1 snRNA is produced in all mammalian cell types, antibiotic resistance genes driven by this promoter would be ideally suited as genetic selection markers. However, although the U1 snRNA gene is transcribed by RNA polymerase II, its native product is not a messenger RNA, but a splicing cofactor. To test whether this promoter could nevertheless produce a functional mRNA, sensitive reporter genes expressing resistance to the antibiotics hygromycin-B and bleomycin were constructed with either the U1 snRNA promoter or the SV40 early promoter. Resistant cell lines could only be obtained with constructs equipped with a functional polyadenylation signal. With the U1 snRNA promoter about three times fewer colonies were obtained than with the SV40 early promoter. Another potential advantage of the U1 snRNA promoter is that, in contrast to the promoters commonly used to express genetic selection markers, the enhancer-like element contained in the U1 snRNA promoter had only a minimal stimulative effect, only detectable with the most sensitive methods, on an adjacent mRNA-producing gene. The U1 snRNA promoter was also capable of expressing bleomycin resistance in the context of a self-inactivating retrovirus vector, whereby it was discovered that the mouse 3T3 cells used in this experiment were 10 times more sensitive to bleomycin than human or hamster cell lines.Abbreviations ble bleomycin resistance gene - Hm hygromycin - hpt hygromycin phosphotransferase gene - neo neomycin (geneticin) phosphotransferase gene     

我国大麦和性花叶病毒（BaMMV）研究初报

本文根据F(ab′)2－ELISA和ISEM实验,结合病毒粒子长度测定,报道了江苏省如东县农科所大麦黄花病毒源（吕种盐辐矮早3）中除存在大麦花花叶病毒（BaYMV）外,还复合感染大麦和性花叶病毒（BaMMV）,这是我国BaMMV的第一次报道。     

乙型肝炎病毒(HBV)体内外对单个核细胞白细胞介素-1和白细胞介素-2诱生活性影响的研究

本研究用PAP法、胸腺细胞增殖法、脾细胞增殖法,分别检测16例体外HBV感染的骨髓单个核细胞与16例慢性乙型肝炎患者体内感染的骨髓单个核细胞(MNCs)中的HBcAg和白细胞介素-1(IL-1)、白细胞介素-2(IL-2)的诱生活性(以△cpm值表示)。结果显示,体外HBV感染组与体内HBV感染组骨髓MNCs中HBcAg检出率分别为50％和43.7％。本实验结果表明,HBV在体外感染骨髓MNCs,且与体内自然感染相符,但光镜下未观察到致细胞病变效应(CPE)。体外感染组与体内感染组IL-I和IL-2活性均较对照组明显下降(P<0.01)。且细胞中HBcAg检出阳性者较阴性者下降更为明显(P<0.01)。IL-1和IL-2诱生活性降低与HBV侵染免疫细胞及其在细胞内复制有密切关系,从而提示,IL-1和IL-2降低可能影响HBV的清除而引起慢性化过程。     

戊型肝炎病毒中国XT—179株全基因组Cdna克隆及其核苷酸和氨基酸序列分析

本文报道对中国新疆东部一起戊型肝炎流行高峰期间,由ＨＥ患者烘便中分离到的型肝炎病毒中国ＸＴ－１７９株进行全基因ｃＤＮＡ克隆、核苷酸和氨基酸序列测定及分析结果。所阐明的ＨＥＶ－ＸＴ－１７９株基因组全序列由７１９４个核苷酸和３＇端的多聚腺嘌呤核苷酸尾组成。四种核苷酸的含量腺嘌呤（Ａ）为１７．０％,胞嘧啶（Ｃ）为３１．９％,鸟嘌呤（Ｇ）为２６．０,尿嘧啶（Ｕ）为２５．１％。Ｇ＋Ｃ含量为５７．９％。全基因     

Generation and effective enrichment of selectable human cytomegalovirus mutants using site-directed insertion of the neo gene

Studies on the biology and function of human cytomegalovirus (HCMV) genes have been hampered by the limited number of viral mutants available for genetic analyses. We have developed a simple procedure to generate and enrich for HCMV recombinants. By inserting the bacterial neo gene, encoding neomycin/kanamycin phosphotransferase, into the large HCMV DNA genome using homologous recombination, selectable mutants of this complex herpesvirus were isolated for the first time. The synthesis of Neo from the viral genome was used to effectively enrich for recombinant viruses (re-viruses) in permissive culture cells grown in the presence of Geneticin (G418). A quick assay for Neo activity in infected cells, based on phosphorylation of kanamycin (Km), was used to easily identify viral recombinants in the process of screening and isolation. This procedure, not used previously to identify re-viruses, proved to be very useful for screening of large numbers of HCMV recombinants. Analysis of re-virus by Southern blotting revealed that the insertion of the marker gene had resulted in the expected deletion of the open reading frames, TRL 13/14 and UL 1–5, of HCMV. Re-virus was stable and showed no differences in growth kinetics as compared to wild-type (wt) virus. The insertion of a selectable marker gene into the HCMV genome and identification of viral recombinants by the Km phosphorylation assay, as presented here, provides the rationale for effective generation, enrichment and stable propagation of HCMV mutants.