Characterization of an HSP70 Cognate Gene Family in Arabidopsis

Analysis of the polypeptide composition of extracts from heat-shocked leaves of Arabidopsis indicated the presence of at least 12 HSP70-related polypeptides, most of which were constitutively expressed. In vitro translation of mRNA from heat-shocked and control leaves indicated that the amount of mRNA encoding four HSP70 polypeptides was increased strongly by heat-shock. Three Arabidopsis genes which exhibit homology to a Drosophila HSP70 gene were cloned. Two of the three genes are arranged in direct orientation approximately 1.5 kilobases apart. The third gene is not closely linked to the other two. Nucleotide sequence analysis of the 5′ regions of the two linked genes revealed that both contain a TATA box, the CAAT motif, and several short sequences which are homologous to the Drosophila heat-shock consensus sequence. The deduced partial amino acid sequence of the open reading frames were 79 and 72% homologous to the corresponding regions of the Drosophila HSP70-cognate and HSP70 sequences, respectively. As with the two maize HSP70 genes which have been characterized, and the Drosophila HSP70-cognate genes, the Arabidopsis genes contained a putative intron in the codon specifying amino acid 72. Analysis of mRNA levels with gene-specific oligonucleotide probes indicated that two of the genes were not expressed or were expressed at very low levels in leaves during normal growth or after heat-shock, whereas the other gene was constitutively expressed. By analogy with the results of similar studies of other organisms, it appears that the three cloned genes are members of a small family which are most closely related to the HSP70-cognate genes found in other species.     

Translational alterations in maize leaves responding to pathogen infection, paraquat treatment, or heat shock : polysome dissociation and accumulation of a 57 kilodalton protein

Translational alterations occur in maize (Zea mays L.) leaves stressed by pathogen infection or herbicide paraquat treatment. These translational changes include: (a) dissociation of large polysomes to small polysomes, monosomes, and subunits; (b) a decreased rate of total protein synthesis; and (c) a reduced synthesis of several proteins by polysomes in vitro. The polysome dissociation was neither due to an extraction artifact nor to degradation of RNA by RNase. The protein patterns of polysomes isolated from leaves inoculated with Bipolaris maydis at 6 to 48 hours showed an increase in the intensity of a 57 kilodalton protein. When inoculated with less virulent pathogens, such as B. zeicola, Exserohilum turcicum, or Colletotrichum graminicola, the protein was accumulated in polysomes of leaves at 24 to 48 hours after inoculation. The 57 kilodalton protein was also accumulated in polysomes of maize leaves responding to heat shock or herbicide paraquat treatments. The purified 57 kilodalton protein reassociated with polysomes isolated from healthy leaves and inhibited polysomal translation in vitro. Since the 57 kilodalton protein is rapidly accumulated in maize polysomes in response to various biological and environmental stresses and may affect protein synthesis, it may be involved in translational regulation of maize leaves during stress response.     

Signal transduction of gamma/delta T cell antigen receptor with a novel mitogenic anti-delta antibody

Human T lymphocytes express either alpha/beta- or gamma/delta-TCR in association with the CD3 complex. We have isolated a mAb, delta TCS1, that immunoprecipitated the gamma/delta-TCR heterodimer from cell lysates of Peer and Molt-13 leukemia cell lines. After dissociation of the gamma- and delta-chains of TCR by treatment with SDS, delta TCS1 specifically immunoprecipitated the delta-chain. This antibody bound to the surface of other gamma/delta-positive T cell lines and clones and was able to stimulate the proliferation of a minor cell population (0.9 to 4.0%) of resting human PBL. Upon binding to gamma/delta-TCR-bearing Molt-13 cells and PBL, delta TCS1 elicited a fura-2 Caa+ signal indicating that the gamma/delta-receptor is functionally similar to the alpha/beta-heterodimer. These data indicate that the delta TCS1 antibody recognizes an epitope on TCR delta-chain and its mitogenic activity should be useful in characterizing the functional properties of human gamma/delta-positive T lymphocytes.     

Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.     

镉对雄性小鼠生精细胞的影响

为了研究镉对小鼠生殖力和生精细胞的作用,本文对氯化镉处理后的雄性小鼠进行了交配实验,以观察其对怀孕率、每窝产仔数及子代性比的影响。测定比较了注射镉后,小鼠成熟精子的总LDH酶和LDH-X酶的活性;还用双向电泳方法分析了成熟精子的蛋白质变化。结果表明,处理组在怀孕率、每窝产仔数及子代性比方面无统计学意义的差异。成熟精子的总LDH活性经镉处理后未发现明显变化,但镉能显著地抑制与精子运动的能量有关的LDH-X酶的活性。双向电泳图谱表示,镉处理后,精子中含量较少的三组蛋白质或消失不见,或发生明显变化。     

九连小蘖悬浮培养细胞生理生化研究 Ⅰ.细胞生长与培养液的电导率和过氧化物酶活性的变化

本文报道了九连小蘖细胞悬浮培养过程中,细胞生长与培养液的电导率、pH值、可溶性糖含量及过氨化物酶活性的变化。实验表明细胞生长曲线与培养液的电导率、可溶性糖含量变化的曲线恰成镜像关系。而细胞生长曲线与培养液的过氨化物酶活性变化的曲线相互平行。从而,可以通过监测培养液的电导率和过氧化物酶活性的变化来了解细胞生长状况,并可作为植物细胞培养过程中生物量增长的参考指标。     

D10S20, a previously unmapped RFLP (OS-3), is located on 10q near D10S4

The locus recognized by the probe OS-3 is assigned to chromosome 10 both by Southern blot analysis of a panel of somatic cell hybrid DNAs and by genetic linkage to markers already assigned to chromosome 10. In Caucasians this probe recognizes a three-allele TaqI RFLP as well as two-allele BanII and RsaI RFLPs which are both in strong linkage disequilibrium with each other and with the TaqI RFLP. The D10S20 locus defined by this probe maps 5.5 cM distal to D10S4 on the long arm of chromosome 10. Because this human clone hybridizes with mouse genomic DNA, it will be useful in comparative mapping studies.     

Genotyping and sequence analysis of apolipoprotein E isoforms

Apolipoprotein E (apoE), a polymorphic plasma protein, is essential for catabolism of lipoproteins by receptor-mediated endocytosis. One of the apoE isoforms (E2) differs in its binding affinity to specific receptors and contributes to variations in lipoprotein metabolism. Diagnosis of apoE isoforms is done by isoelectric focusing, but it is hindered by various degrees of post-translational sialylation of the apoE protein. Electrophoretically silent structural variations may also escape detection by this technique. We describe a method for genotyping apoE based on hybridization of allele-specific oligonucleotides with enzymatically amplified genomic DNA, which permits unambiguous diagnosis of six common apoE phenotypes within 24 h. Among 100 E2 alleles present in 81 unrelated individuals genotyped by this technique, we found two rare structural mutants of apoE in addition to the common E2 form, E2(158Arg----Cys). Automated sequencing of amplified DNA identified the rare mutants as E2(136Arg----Ser) and E2(145Arg----Cys). The genotypic method may complement or even replace isoelectric focusing for routine determination of apoE phenotypes and for identification of rare structural variants.     

Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene

Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus.