与胎儿珠蛋白基因持续表达有关的(A_γδβ)°—地贫纯合子及杂合子的分子鉴别

我们分子鉴别了一个缺失型中国(A_γδβ)°-地贫家系。先证者为这一缺失的纯合子,具有中度贫血症状。家系的另五个成员均为这一缺失的杂合子,其胎儿血红蛋白(HbF)为16—21％,接近或达到HPFH杂合子的HbF水平,并且几乎不表现贫血症状。限制性内切酶图谱分析证明了β-珠蛋白基因簇内的DNA顺序缺失,缺失的5′端点位于Aγ基因IVSⅡ内,3′端点在β-珠蛋白基因下游区远端,距HPFH-2的3′缺失端点上游区约11kb。缺失的总长度约为80kb。本文讨论了这一缺失导致胎儿血红蛋白在成人中持续活跃表达的可能机制。     

基因定点诱变删除及天然α型心钠素的分泌型表达

用基因定点诱变技术,删除了pO_1α ANF表达质粒中的33对碱基,使人α型心钠素结构基因直接与大肠杆菌分泌型表达质粒pIN-Ⅲ-OmPA中的信号肽酶切位点编码区相连,构成天然人α型心钠素的表达质粒pANF,在IPTG诱导下表达28肽的天然人α型心钠素。纯化后的表达产物具有天然心钠素的放免活性和很强的舒张血管的生物活性。     

Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: Module evolution of the filamentous phage genome

Summary The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism. Offprint requests to: R.N.H. Konings     

Conformational properties of oxytocin in dimethyl sulfoxide solution: NMR and restrained molecular dynamics studies.

The conformation of oxytocin, the neurohypophyseal nonapeptide hormone, in solution in deuterated dimethyl sulfoxide has been determined by 1H-nmr. The structural determination is based on the experimental data set of nuclear Overhauser effect restraints. Obtained after the restrained molecular dynamics simulation on an initial structure of extended conformation, five resultant structures satisfy the experimental restraints well. These structures resemble that of the crystal structure of deamino-oxytocin, an analogue of oxytocin, in terms of a close correlation observed both at two beta-turn regions of the 20-membered tocin ring and at the tripeptide tail end. Based on this comparison and analysis of restrained molecular dynamics trajectories, we found that, although the turns are stabilized by the formation of hydrogen bonds, the oxytocin molecule possesses a slight twist in DMSO solution relative to the orientation of deamino-oxytocin in the crystalline state. Analyses of oxytocin conformation indicate that the tripeptide tail is more flexible than the tocin ring.     

Early stages of formation and dispersal of the temperate flora in the Boreal Region

Budantsev, L. Yu. (Komarov Botanical Institute, Prof. Popov Str. 2, 197376 St. Petersburg, Russia). Early stages of formation and dispersal of the temperate flora in the Boreal Region. Bot. Rev.58(1): 1–48, 1992.—The thesis of this review is that, as stated as early as 1908 by V. L. Komarov, the composition of a flora can be understood only as a process, or separate stage, in the context of migration in time and space of various floristic assemblages and their isolation, as induced by transformation of continental and ocean shapes, changes in climate, and the environment as a whole. Thus the formation of geofloras of the past was influenced by gradually changing environments that determined the spread, patterning, and spatial differentiation of floras and their evolution. Parallel to the more commonly-seen names of eras—Paleozoic, Mesozoic, and Cenozoic—we can speak of the Paleophytic, Mesophytic, and Cenophytic eras. Eras defined in these two ways (by faunistic or by floristic criteria) do not completely coincide. Generally, changes in the flora have, necessarily, preceded changes in the fauna. It is the Cenophytic with which this review is mostly concerned, the era of Angiosperm dominance. The movement of early subtropical and warm temperate floras in the Early Cenophytic, followed by temperate or even boreal floras, as the climate changes, is traced in detail. The regions discussed most fully are the Boreal-Atlantic and Boreal-Pacific, with emphasis on the Angaro-Beringian flora. The disappearance of archaic forms (e.g., cycadophytes) and the gradual predominance of angiosperms is documented. The movements of the floral assemblages in response to environmental changes are mapped and described. The early development and diversification of the boreal temperate flora is considered to have taken place mainly in Angaro-Beringia, associated with the invasive migration of tropical angiosperms from southeastern Asia.     

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.     

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.     

Active site conformation in myoglobin as determined by X-ray absorption spectroscopy

X-ray absorption fine structure experiments were performed to study structural and dynamic aspects of the active site of various forms of myoglobin. The structures determined for deoxyMb, MbCO, and MbO2 are consistent with the structure established by X-ray absorption fine structure experiment and X-ray crystallography. The first shell of ferrous MbNO determined contains 5 nitrogens located at 2.02 A and a short NO bond length of 1.76 A. This study focuses on the change of the XAFS Debye-Waller factor with temperature, which is a measure of thermal and static disorder. It was found that the changes of Debye-Waller factor with temperature for the Mb proteins, except deoxyMb, are consistent with a simple Einstein model, in which a single frequency was assumed for the bond stretching modes. In contrast, the temperature dependence of deoxyMb cannot be fitted to the Einstein model and a large disorder was found at low temperatures, which indicates the existence of conformational substates of the active site.     

Modulation of respiratory responses to carotid sinus nerve stimulation by brain hypoxia.

This study examines the effect of progressive isocapnic CO hypoxemia on respiratory afterdischarge and the phrenic neurogram response to supramaximal carotid sinus nerve (CSN) stimulation. Twelve anesthetized, vagotomized, peripherally chemodenervated, ventilated cats with blood pressure controlled were studied. During isocapnic hypoxemia, the amplitude of the phrenic neurogram was progressively depressed. In contrast, the increase in peak phrenic amplitude produced by CSN stimulation was unchanged, suggesting that the central respiratory response to CSN stimulation is unaffected by progressive hypoxemia. The time constant of respiratory afterdischarge (tau) was calculated from best-fit plots of phrenic amplitude vs. time after cessation of CSN stimulation. Under control conditions the value of tau was 57.7 +/- 3 (SE) s (n = 12). During progressive isocapnic hypoxemia, tau decreased as a linear function of arterial O2 content (CaO2) such that a 40% reduction of CaO2 resulted in a 48% reduction in tau. This reduction of respiratory afterdischarge may contribute to the genesis of periodic breathing during hypoxia.     

Cloning and sequencing of a cDNA encoding the Rieske iron-sulfur protein of bovine heart mitochondrial ubiquinol-cytochrome c reductase

Two cDNA clones encoding bovine heart mitochondrial Rieske iron-sulfur protein were obtained by immunological screening of a bovine heart cDNA expression library in lambda gt11 with antiserum directed against Rieske iron-sulfur protein isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase. The cDNA inserts were 1005 and 1100 base pairs with an open reading frame of 807 base pairs which encoded a 196-amino acid mature Rieske iron-sulfur protein and a 73-amino acid presequence. The amino acid sequence of Rieske iron-sulfur protein deduced from nucleotide sequencing is the same as that obtained from protein sequencing except at residues #73 and #191 which are Ser and Asp instead of Ala and Gly, respectively.