Vitamin K-dependent blood coagulation proteins form hetero-dimers

The later stages of the blood coagulation cascade are characterized by the presence of vitamin K-dependent proteins and their involvement in membrane-bound, multi-protein converting complexes with an essential requirement for calcium ions. Specific interactions between zymogens and activating enzymes have not yet been identified. Here we describe a crystallographic study of prothrombin fragment 1 (residues 1-156 of prothrombin) which indicates that vitamin K-dependent coagulation proteins have specific association sites that allow them to form hetero-dimers. The calcium-induced formation of a hetero-dimer between fragment 1 and factor X is demonstrated by cross-linking. Such hetero-dimers of vitamin K-dependent proteins could be significant in the coagulation system.     

Tissue-specific and reversible RNA interference in transgenic mice

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.     

Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor

DNA clones specifying the murine granulocyte-macrophage colony stimulating factor have been isolated. This haematopoietic growth factor is encoded by a unique gene specifying a messenger RNA of 1,200 nucleotides and a polypeptide of 118 amino acids. It bears no structural similarity to the functionally related factor, interleukin-3, described recently.     

Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development

To rapidly identify genes required for early vertebrate development, we are carrying out a large-scale, insertional mutagenesis screen in zebrafish, using mouse retroviral vectors as the mutagen. We will obtain mutations in 450 to 500 different genes--roughly 20% of the genes that can be mutated to produce a visible embryonic phenotype in this species--and will clone the majority of the mutated alleles. So far, we have isolated more than 500 insertional mutants. Here we describe the first 75 insertional mutants for which the disrupted genes have been identified. In agreement with chemical mutagenesis screens, approximately one-third of the mutants have developmental defects that affect primarily one or a small number of organs, body shape or swimming behavior; the rest of the mutants show more widespread or pleiotropic abnormalities. Many of the genes we identified have not been previously assigned a biological role in vivo. Roughly 20% of the mutants result from lesions in genes for which the biochemical and cellular function of the proteins they encode cannot be deduced with confidence, if at all, from their predicted amino-acid sequences. All of the genes have either orthologs or clearly related genes in human. These results provide an unbiased view of the genetic construction kit for a vertebrate embryo, reveal the diversity of genes required for vertebrate development and suggest that hundreds of genes of unknown biochemical function essential for vertebrate development have yet to be identified.     

Independent rate and temporal coding in hippocampal pyramidal cells

In the brain, hippocampal pyramidal cells use temporal as well as rate coding to signal spatial aspects of the animal's environment or behaviour. The temporal code takes the form of a phase relationship to the concurrent cycle of the hippocampal electroencephalogram theta rhythm. These two codes could each represent a different variable. However, this requires the rate and phase to vary independently, in contrast to recent suggestions that they are tightly coupled, both reflecting the amplitude of the cell's input. Here we show that the time of firing and firing rate are dissociable, and can represent two independent variables: respectively the animal's location within the place field, and its speed of movement through the field. Independent encoding of location together with actions and stimuli occurring there may help to explain the dual roles of the hippocampus in spatial and episodic memory, or may indicate a more general role of the hippocampus in relational/declarative memory.     

Integration target site selection for retroviruses and transposable elements

When a retrovirus infects a cell, its RNA genome is reverse transcribed into a double-stranded DNA, which is then permanently integrated into the host chromosome. Integration is one of the essential steps in the retroviral life cycle. Many transposable elements also move around and integrate into the host genome as part of their life cycle, some through RNA intermediates and some through 'cut and paste' mechanisms. Integration of retroviruses and transposable elements into 'sensitive areas' of the genome can cause irreparable damage. On the other hand, because of their ability to integrate permanently, and the relatively efficient rates of transgenesis, retroviruses and transposable elements are widely used as gene delivery tools in basic research and gene therapy trials. Recent events in gene therapy treatments for X-linked severe combined immunity deficiencies (X-SCID) have highlighted both the promise and some of the risks involved with utilizing retroviruses. Nine of 11 children were successfully treated for X-SCID using a retrovirus carrying the gene mutated in this disease. However, later two of these children developed leukemias because of retroviral integrations in the putative oncogene LMO2 [1]. A third child has also been demonstrated to have an integration in LMO2, but is as of yet nonsymptomatic [2]. It is a bit difficult to explain the high frequency of integrations into the same gene using a random model of retroviral integration, and there has been evidence for decades that retroviral integrations may not be random. But the data were somewhat limited in their power to determine the precise nature of the integration biases. The completion of the human genome sequence coupled with sensitive polymerase chain reaction techniques and an ever-decreasing cost of sequencing has given a powerful new tool to the study of integration site selection. In this review, we describe the findings from several recent global surveys of target site selection by retroviruses and transposable elements, and discuss the possible ramifications of these findings to both mechanisms of action and to the use of these elements as gene therapy vectors.     

Rapid generation of region specific probes by chromosome microdissection and their application.

The strategy presented here to identify unequivocally cryptic chromosomal rearrangements has relevance to both prenatal and postnatal cytogenetic analysis as well as the analysis of tumour-associated chromosome rearrangements. Microdissection and in vitro amplification of specific chromosomal regions are performed, followed by labelling for fluorescent in situ hybridization (FISH) to normal metaphase chromosomes (Micro-FISH). Micro-FISH probes have been used successfully to determine the derivation of chromosome segments unidentifiable by standard chromosome banding analysis. Micro-FISH probes (created in less than 24 hours) now make it possible to identify explicitly the chromosome constitution of virtually all cytologically visible chromosome rearrangements.     

甘肃民勤盆地人类活动对生态环境的影响及其生态环境变化对人类的反作用研究--民勤盆地社会调查结果分析

主要根据民勤盆地的社会调查资料分析了甘肃省河西地区石羊河流域下游民勤盆地近50年来人类活动对生态环境的影响以及生态环境变化对人类的反作用,并着重分析了当地人对当地生态环境变化及其原因的理解与认识.认为民勤盆地存在环境恶化趋势.该地区地下水位迅速下降,沙漠化、土壤盐渍化、植被衰退过程加快,这些生态环境变化对人类的生产、生活造成了重大而深刻的影响,并对人类身体健康造成了持久的危害.