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71.
R J Siezen E Wu E D Kaplan J A Thomson G B Benedek 《Journal of molecular biology》1988,199(3):475-490
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. 相似文献
72.
73.
Genotyping and sequence analysis of apolipoprotein E isoforms 总被引:24,自引:0,他引:24
M Emi L L Wu M A Robertson R L Myers R A Hegele R R Williams R White J M Lalouel 《Genomics》1988,3(4):373-379
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. 相似文献
74.
Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene 总被引:12,自引:0,他引:12
D T Drayna R A Hegele P E Hass M Emi L L Wu D L Eaton R M Lawn R R Williams R L White J M Lalouel 《Genomics》1988,3(3):230-236
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. 相似文献
75.
76.
Simple spike of cerebellar Purkinje cells (PC-SS) was recorded with microelectrode. In the NCCVF (normalized cross-covariance function) histogram, spontaneous PC-SS does not show obvious peak. When the saphenous nerve is stimulated at lower intensities, which elicits the A-fiber input only, the discharge response (A-CED) consists of an early component with a latency of 16.7 +/- 0.9 ms and a late component with a latency of 270.8 +/- 12.8 ms. After A-fibers are blocked selectively by polarizing current, the stimulation at a suprathreshold strength for C-fiber evokes a characteristic response (C-CED) with a latency of 142.4 +/- 4.3 ms. However, the C-CED can not be evoked by the inputs of A- and C-fiber simultaneously. In NPSDF histogram, the spontaneous activities of PC-SS can be divided into two groups, the high and the low peak group. The high peak group (n = 15) has a peak energy value of 15.7 +/- 4.7 x 10(-3) and peak frequency of 4.07 +/- 1.69 Hz. A-fiber input causes an increase of the peak value, while C-fiber input causes a decrease. The low peak group (n = 16) has a peak energy value 8.4 +/- 1.4 x 10(-3) and peak frequency of 3.67 +/- 2.90 Hz. Both A-fiber and C-fiber inputs cause an increase of the peak value, but the effect of A-fiber input was more prominent. The results show that the pure C-fiber input can reach the cerebellar PC and elicit characteristic simple spike response. 相似文献
77.
78.
利用透射电镜技术研究了生长在海南岛的热带落叶树降香黄檀(Dalbegia odorifera T.Chen)1—2年生枝条着叶期和无叶期次生韧皮部筛分子的超微结构,并就这两个时期的筛分子进行了比较。着叶期每个成熟筛分子内有一个带尾的纺锤形P-蛋白质体,主体由稠密而散乱的P-蛋白质细纤维组成,尾部呈结晶状;筛分子具有横向端壁和单筛板,在邻近筛板处,细胞壁向筛分子腔内形成明显的突起。无叶期仍然保持着与着叶期大致相同厚度的有功能韧皮部,筛分子具有正常的原生质体,P-蛋白质和筛板孔的结构也与着叶期的相同,但筛分子内有较多的淀粉粒和囊泡。 相似文献
79.
To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations
Adh
alcohol dehydrogenase
- BAP
benzylaminopurine
- bp
base pair(s)
- GUS
-glucuronidase
- Kb
kilobase(s)
- MS
Murashige and Skoog's medium
- PCR
polymerase chain reaction 相似文献
80.
Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line. 总被引:20,自引:16,他引:4 下载免费PDF全文
The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV. 相似文献