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1.
Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.  相似文献
2.
A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.  相似文献
3.
Shock and tissue injury induced by recombinant human cachectin   总被引:286,自引:0,他引:286  
Cachectin (tumor necrosis factor), a protein produced in large quantities by endotoxin-activated macrophages, has been implicated as an important mediator of the lethal effect of endotoxin. Recombinant human cachectin was infused into rats in an effort to determine whether cachectin, by itself, can elicit the derangements of host physiology caused by administration of endotoxin. When administered in quantities similar to those produced endogenously in response to endotoxin, cachectin causes hypotension, metabolic acidosis, hemoconcentration, and death within minutes to hours, as a result of respiratory arrest. Hyperglycemia and hyperkalemia were also observed after infusion. At necropsy, diffuse pulmonary inflammation and hemorrhage were apparent on gross and histopathologic examination, along with ischemic and hemorrhagic lesions of the gastrointestinal tract, and acute renal tubular necrosis. Thus, it appears that a single protein mediator (cachectin) is capable of inducing many of the deleterious effects of endotoxin.  相似文献
4.
Complementary DNA sequencing: expressed sequence tags and human genome project   总被引:227,自引:0,他引:227  
Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.  相似文献
5.
Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with which clones can be generated is high enough to allow the construction of comprehensive libraries from the genomes of higher organisms. By offering a tenfold increase in the size of the DNA molecules that can be cloned into a microbial host, this system addresses a major gap in existing experimental methods for analyzing complex DNA sources.  相似文献
6.
7.
Microfilaments in cellular and developmental processes   总被引:184,自引:0,他引:184  
In our opinion, all of the phenomena that are inhibited by cytochalasin can be thought of as resulting from contractile activity of cellular organelles. Smooth muscle contraction, clot retraction, beat of heart cells, and shortening of the tadpole tail are all cases in which no argument of substance for alternative causes can be offered. The morphogenetic processes in epithelia, contractile ring function during cytokinesis, migration of cells on a substratum, and streaming in plant cells can be explained most simply on the basis of contractility being the causal event in each process. The many similarities between the latter cases and the former ones in which contraction is certain argue for that conclusion. For instance, platelets probably contract, possess a microfilament network, and behave like undulating membrane organelles. Migrating cells possess undulating membranes and contain a similar network. It is very likely, therefore, that their network is also contractile. In all of the cases that have been examined so far, microfilaments of some type are observed in the cells; furthermore, those filaments are at points where contractility could cause the respective phenomenon. The correlations from the cytochalasin experiments greatly strengthen the case; microfilaments are present in control and "recovered" cells and respective biological phenomena take place in such cells; microfilaments are absent or altered in treated cells and the phenomena do not occur. The evidence seems overwhelming that microfilaments are the contractile machinery of nonmuscle cells. The argument is further strengthened if we reconsider the list of processes insensitive to cytochalasin (Table 2). Microtubules and their sidearms, plasma membrane, or synthetic machinery of cells are presumed to be responsible for such processes, and colchicine, membrane-active drugs, or inhibitors of protein synthesis are effective at inhibiting the respective phenomena. These chemical agents would not necessarily be expected to affect contractile apparatuses over short periods of time, they either do not or only secondarily interfere with the processes sensitive to cytochalasin (Table 1). It is particularly noteworthy in this context that microtubules are classed as being insensitive to cytochalasin and so are not considered as members of the "contractile microfilament" family. The overall conclusion is that a broad spectrum of cellular and developmental processes are caused by contractile apparatuses that have at least the common feature of being sensitive to cytochalasin. Schroeder's important insight (3) has, then, led to the use of cytochalasin as a diagnostic tool for such contracile activity: the prediction is that sensitivity to the drug implies presence of some type of contractile microfilament system. Only further work will define the limits of confidence to be placed upon such diagnoses. The basis of contraction in microfilament systems is still hypothetical. Contraction of glycerol-extracted cells in response to adenosine triphosphate (53), extraction of actin-like or actomyosin-like proteins from cells other than muscle cells (54), and identification of activity resembling that of the actomyosin-adenosine triphosphatase system in a variety of nonmuscle tissues (40, 54) are consistent with the idea that portions of the complex, striated muscle contractile system may be present in more primitive contractile machinery. In the case of the egg cortex, calcium-activated contractions can be inhibited by cytochalasin. If, as seems likely, microfilaments are the agents activated by calcium, then it will be clear that they have the same calcium requirement as muscle. Biochemical analyses of primitive contractile systems are difficult to interpret. Ishikawa's important observation (31), that heavy meromyosin complexes with fine filaments oriented parallel to the surface of chondrocytes and perpendicular to the surface of intestinal epithelial cells, implies that both types of filaments are "actin-like" in this one respect. Yet, it is very likely that these actin-like filaments correspond respectively to the cytochalasin-insensitive sheath of glial and heart fibroblasts and the core filaments of oviduct microvilli. No evidence from our studies links contractility directly to these meromyosin-binding filaments. Apart from this problem, activity resembling that of the myosin-adenosine triphosphatase has been associated with the microtubule systems of sperm tails and cilia (55), but those organelles are insensitive to cytochalasin in structure and function. Clearly, a means must be found to distinguish between enzymatic activities associated with microfilament networks, microfilament bundles, microtubules, and the sheath filaments of migratory cells. Until such distinctions are possible, little of substance can be said about the molecular bases of primitive contractile systems. Three variables are important for the control of cellular processes dependent upon microfilaments: (i) which cells of a population shall manufacture and assemble the filaments; (ii) where filaments shall be assembled in cells; and (iii) when contractility shall occur. With respect to distribution among cells, the networks involved in cell locomotion are presumed to be present in all cells that have the potential to move in cell culture. In this respect, the networks can be regarded as a common cellular organelle in the sense that cytoplasmic microtubules are so regarded. In some developing systems, all cells of an epithelium possess microfilament bundles (7, 13), whereas, in others, only discrete subpopulations possess the bundles (5, 6). In these cases the filaments can be regarded as being differentiation products associated only with certain cell types. These considerations may be related to the fact that microfilament networks are associated with behavior of individual cells (such as migration, wound healing, and cytokinesis), whereas the bundles are present in cells that participate in coordinated changes in shape of cell populations. With respect to placement in cells, two alternatives are apparent, namely, localized or ubiquitous association with the plasma membrane. Microfilament bundles of epithelial cells are only found extending across the luminal and basal ends of cells. In this respect they contrast with desmosomal tonofilaments and with microtubules, each of which can curve in a variety of directions through the cell. The strict localization of microfilament bundles probably rests upon their association with special junctional complex insertion regions that are only located near the ends of cells. In the case of mitotically active cells, the orientation of the spindle apparatus may determine the site at which the contractile ring of microfilaments will form (4, 56); this raises the question of what sorts of cytoplasmic factors can influence the process of association between filament systems and plasma membranes. In contrast to such cases of localized distribution, contractile networks responsible for cell locomotion are probably found beneath all of the plasma membrane, just as the network of thrombosthenin may extend to all portions of the periphery of a blood platelet. This ubiquitous distribution probably accounts for the ability of a fibroblast or glial cell to establish an undulating membrane at any point on its edge, or of an axon to form lateral microspikes along its length. The third crucial aspect of control of these contractile apparatuses involves the choice of when contraction shall occur (and as a corollary the degree or strength of contraction that will occur). In the simplest situation, contraction would follow automatically upon assembly of the microfilament bundles or networks. In cleavage furrows of marine embryos (4), for instance, microfilaments are seen beneath the central cleavage furrow and at its ends, but not beyond, under the portion of plasma membrane that will subsequently become part of the furrow. This implies that the furrow forms very soon after the contractile filaments are assembled in the egg cortex. In other cases, microfilaments are apparently assembled but not in a state of (maximal?) contraction. Thus, networks are seen along the sides of migratory cells, although such regions are not then active as undulating membrane organelles. Similarly, microfilament bundles occur in all epithelial cells of the salivary gland (13), or pancreatic anlage (7), although only the ones at discrete points are thought to generate morphogenetic tissue movements. Likewise, bundles begin to appear as early as 12 hours after estrogen administration to oviduct, although visible tubular gland formation does not start until 24 to 30 hours. Finally, streaming in plant cells can wax and wane, depending upon external factors such as auxin (57). All of these cases imply a control mechanism other than mere assembly of the microfilament systems and even raise the possibility that within one cell some filaments may be contracting while others are not. In discussing this problem, it must be emphasized that different degrees of contraction or relaxation cannot as yet be recognized with the electron microscope. In fact, every one of the cases cited above could be explained by contraction following immediately upon some subtle sort of "assembly." Inclusive in the latter term are relations between individual filaments, relations of the filaments and their insertion points on plasma membrane, and quantitative alterations in filament systems. Furthermore, the critical role of calcium and high-energy compounds in muscle contraction suggest that equivalent factors may be part of primitive, cytochalasinsensitive systems. The finding that calcium-induced contraction in the cortex of eggs is sensitive to cytochalasin strengthens that supposition and emphasizes the importance of compartmentalization of cofactors as a means of controlling microfilaments in cells.  相似文献
8.
Genes and operons that encode bacterial virulence factors are often subject to coordinate regulation. These regulatory systems are capable of responding to various environmental signals that may be encountered during the infectious cycle. For some pathogens, proteins that mediate sensory transduction and virulence control are similar to components of other bacterial information processing systems. Understanding the molecular mechanisms governing global regulation of pathogenicity is essential for understanding bacterial infectious diseases.  相似文献
9.
Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas   总被引:175,自引:0,他引:175  
Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.  相似文献
10.
Familial cancer syndromes have helped to define the role of tumor suppressor genes in the development of cancer. The dominantly inherited Li-Fraumeni syndrome (LFS) is of particular interest because of the diversity of childhood and adult tumors that occur in affected individuals. The rarity and high mortality of LFS precluded formal linkage analysis. The alternative approach was to select the most plausible candidate gene. The tumor suppressor gene, p53, was studied because of previous indications that this gene is inactivated in the sporadic (nonfamilial) forms of most cancers that are associated with LFS. Germ line p53 mutations have been detected in all five LFS families analyzed. These mutations do not produce amounts of mutant p53 protein expected to exert a trans-dominant loss of function effect on wild-type p53 protein. The frequency of germ line p53 mutations can now be examined in additional families with LFS, and in other cancer patients and families with clinical features that might be attributed to the mutation.  相似文献
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