生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

从我国成人手足口病患者疱液中首次分离出肠道病毒71型

本文报道了从我国手足口病(HFMD)患者疱液中肠道病毒71(E71)型H株的分离和鉴定。该株病毒可在原代人胚肺(HEL)细胞、MA104细胞、BSC细胞中生长繁殖,导致典型的肠道病毒CPE出现。将H株接种乳鼠后出现肢体麻痹、第6天开始死亡。电镜下可见感染H株的BSC细胞胞浆中出现大量的结晶状排列的成熟病毒颗粒,直径约25nm。患者双份血清中有对H株4倍增高的中和抗体存在。采用100抗体单位的抗Cox A5、7、9、16、E70、E71抗体和50抗体单位的LBM组合血清A-H以及抗E71BrCr株MeAb P27对H株进行中和试验时,H株可被抗E71血清和MeAb P27所中和。抗E71抗体对H株的最低有效中和作用为1.6抗体单位,MeAb P27对H株的有效中和作用是64抗体单位。其它血清则无此中和作用。然而,在鉴定过程中发现,高滴度的抗Cox A16抗体(200抗体单位以上)也显出有中和H株的作用,提示我们所分离的H株含有与Cox A16的型间共同抗原。     

^60Co—γ射线诱导的小麦T型雄性不育系育性恢复突变

用~(60)Co-γ射线诱导小麦T型雄性不育系T小偃4号A、T郑引1号A均获得了农艺性状优良、恢复力强的恢复系。育性恢复突变是涉及一个或少数几个位点核基因的显性突变。利用性状优良的T型不育系诱导育性恢复突变是选育恢复系和发掘恢复源的有效途径。     

中棉×戴维逊氏棉胚胎发育的研究

以棉花栽培种中棉作母本,野生种戴维逊氏棉作父本进行杂交试验,并用中棉自交作对照,比较研究了杂交情况下花粉粒的萌发、花粉管的生长、受精作用及胚和胚乳的发育过程,得到以下结果:(1)中棉×戴维逊氏棉花粉粒的萌发及花粉管在异己花柱中的生长基本正常,有花粉管胚珠的频率约20％,为中棉自交的1/4左右;(2)在杂交情况下,有花粉管进入的胚珠基本上能实现受精;(3)杂种胚乳在授粉后7天发育异常,11天开始解体,16天才有部分胚珠的胚乳开始形成细胞壁;(4)杂种胚不分化或畸形分化,在授粉后11—22天坏死。     

PUC衍生质粒的一个低拷贝数突变型

本文报道大肠杆菌的ColE1类似质粒的一个低拷贝数突变型。从载体质粒pUC4衍生的重组质粒pPGVT3在大肠杆菌宿主DF2145中是不稳定的,以pPGVT3转化DF2145时在4o℃培养得不到转化子。用诱发点突变的羟胺体外处理pPGVF3质粒DNA,得到一个稳定性提高了的突变质粒pPGVT3HA,突变的位置被确定在质粒的pUC部分,突变降低了pUC及其衍生质粒的拷贝数。文中对质粒的稳定性与拷贝数的关系作了讨论。     

Assay of L-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.     

Effects of acetylene on hydrogenases from the sulfate reducing and methanogenic bacteria

The effect of acetylene on the activity of the three types of hydrogenase from the anaerobic sulfate reducing bacteria has been investigated. The (Fe) hydrogenase is resistant to inhibition by acetylene while the nickel-containing hydrogenases are inhibited by acetylene with the (NiFe) hydrogenase being 10-50 fold more sensitive than the (NiFeSe) hydrogenase. In addition the Ni(III) EPR signal (g approximately 2.3) of the "as isolated" (NiFe) hydrogenase was significantly decreased in intensity upon exposure to acetylene.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

EPR-detectable redox centers of the periplasmic hydrogenase from Desulfovibrio vulgaris

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) belongs to the category of [Fe] hydrogenase which contains only iron-sulfur clusters as its prosthetic groups. Amino acid analyses were performed on the purified D. vulgaris hydrogenase. The amino acid composition obtained compared very well with the result derived from the nucleotide sequence of the structural gene (Voordouw, G., Brenner, S. (1985) Eur. J. Biochem. 148, 515-520). Detailed EPR reductive titration studies on the D. vulgaris hydrogenase were performed to characterize the metal centers in this hydrogenase. In addition to the three previously observed EPR signals (namely, the "isotropic" 2.02 signal, the rhombic 2.10 signal, and the complex signal of the reduced enzyme), a rhombic signal with resonances at the g-values of 2.06, 1.96, and 1.89 (the rhombic 2.06 signal) was detected when the samples were poised at potentials between 0 and -250 mV (with respect to normal hydrogen electrode). The midpoint redox potentials for each of the four EPR-active species were determined, and the characteristics of each EPR signal are described. Both the rhombic 2.10 and 2.06 signals exhibit spectral properties that are distinct from a ferredoxin-type [4Fe-4S] cluster and are proposed to originate from the same H2-binding center but in two different conformations. The complex signal of the reduced hydrogenase has been shown to represent two spin-spin interacting ferredoxin-type [4Fe-4S]1+ clusters (Grande, H. J., Dunham, W. R., Averill, B., Van Dijk, C., and Sands, R. H. (1983) Eur. J. Biochem. 136, 201-207). The titration data indicated a strong cooperative effect between these two clusters during their reduction. In an effort to accurately estimate the number of iron atoms/molecule of hydrogenase, plasma emission and chemical methods were used to determine the iron contents in the samples; and four different methods, including amino acid analysis, were used for protein determination. The resulting iron stoichiometries were found to be method-dependent and vary over a wide range (+/- 20%). The uncertainties involved in the determination of iron stoichiometry are discussed.     

Endurance at intermediate swimming speeds of Atlantic mackerel, Scomber scombrus L., herring, Clupea harengus L., and saithe, Pollachius virens L.

Endurance and swimming speed were measured in mackerel, herring and saithe when they were induced by the optomotor response to swim at prolonged speeds along a 28-m circular track through still water in a 10-m diameter gantry tank. The maximum sustained swimming speed ( U _ms was measured as body lengths per second ( b.l.s ⁻¹) for each species and for saithe of different size groups. Herring with U _ms of 4.06 b.l.s ⁻¹ (25.3 cm, 13.5°C) were the fastest, mackerel U _ms was 3.5 b.l.s ¹ (33 cm, 11.7°C) and saithe (14.4°C) showed a size effect where U _ms at 25 cm was 3.5 b.l.s ¹ and at 50 cm 2.2 b.l.s ¹. When swimming at speeds higher that U _ms, all three species showed reduced endurance as speed increased. How the curved track reduces the swimming speed is discussed.