Rhodes and Konieczny, 1989) and MyoD (Myod1 -Mouse Genome Informatics) (Davis et al., 1987). These are members of the basic helix-loop-helix (bHLH) family of transcription factors (reviewed by Arnold and Braun, 2000). Myf5 is the first The initiation of skeletal muscle development in the mouse embryo is strictly associated with the expression of the muscle-specific transcription factor Myf5, the first of four myogenic regulatory factors (MRFs) to be expressed in muscle progenitors, and ablation of the Myf5 gene prevents myogenesis. The complex spatiotemporal expression pattern of Myf5 depends on many discrete regulatory elements that are dispersed over long distances throughout the gene locus. These multiple control modules act differently in the various muscle precursor populations, presumably in response to diverse signals that control myogenesis. A potent enhancer region regulating Myf5 expression in limb muscles and somites has been identified previously at -58/-48 kb upstream of the transcriptional start site (Hadchouel et al., 2000). Here, we focus on the physical and functional dissection of this control region. We demonstrate that a conserved sequence of 270 bp located around -57 kb is required and sufficient to drive Myf5 expression in limbs and to maintain it in somites. A second enhancer nearby is responsible for Myf5 transcription in occipital/cranial somites. This enhancer activity also directs expression accurately to the myotome, preventing ectopic expression in the dermomyotome during the second phase of Myf5 gene activation in somites.Our data suggest that the enhancer identified here collaborates with other somitic enhancers to ensure correct myotomal Myf5 expression. Moreover, it constitutes an important element that mediates somitic expression after the initial and transient Myf5 activation through a previously described sonic hedgehog-dependent early epaxial enhancer.
Analysis of 505 cases history of patients among men with viral hepatitis demonstrates that HBV infected patients represent 68.9% of the total and that a non-parenteral rate of transmission is the most likely means of hepatitis B infection. Saliva and serum testing for the presence of specific HBV markers (HBsAg, HBeAg and HBV DNA) at different phases of the infection process were carried out to review the diagnostic and epidemiological value of saliva samples from patients with acute viral hepatitis B. The frequency of HBsAg detection by Enzyme Immune Assay (EIA) in saliva of patients in acute period was found to correlate with the frequency of its detection in serum. In early convalescence the frequency of detection of that antigen in serum (59.5% of patients) was significantly higher than in saliva (23.8%) (P < 0.001). The frequencies of HBeAg detection by EIA in saliva samples was significantly higher than that in serum samples in both acute phase (84.3% and 28.1% of patients, respectively) and in early convalescence (56.2% and 3.1% of patients, respectively). The study of frequencies of detection of these antigens in the dynamics of the disease up to the total recovery of patients (observations were carried out for the period of 60 days and longer) showed that in most patients there was a faster disappearance HBsAg from saliva than from serum. By the end of second month this antigen was detected in saliva of only 8.3% of patients whereas in serum in the same period HBsAg was detected in 33.3% of patients. HBeAg became undetectable in blood whereas HBs-antigenemia was still pronounced, and a month after the beginning of the disease it was not found in serum specimens. In saliva, HBeAg was detected in 95.8% of patients observed directly after admission. A month after the beginning of the disease it was detected in saliva of 66.7% of patients and, by the end of observation period, in 12.5% of patients recovered from viral hepatitis. HBV DNA revealed by PCR in saliva and serum of HBV-infected patients was detected in acute period not only in serum (84.6% of cases) but also in saliva (46.2% of cases). The data illustrate the diagnostic value of saliva and point to the possible role of saliva as a source of HBV infection.
Skeletal muscle development in the vertebrate embryo critically depends on the myogenic regulatory factors (MRFs) including MRF4 and Myf5. Both genes exhibit distinct expression patterns during mouse embryogenesis, although they are genetically closely linked with multiple regulatory elements dispersed throughout the common gene locus. MRF4 has a biphasic expression profile, first in somites and later in foetal skeletal muscles. Here, we demonstrate by transgenic analysis that elements within a 7.5-kb promoter fragment of the MRF4 gene are sufficient to drive the embryonic wave of expression very similar to the endogenous gene in somites of mouse embryos. In contrast, a 3-kb fragment of the proximal promoter fails to support expression in the myotome, suggesting that essential cis-acting elements are located between -7.5 and -3 kb upstream of MRF4. Further analysis of this sequence delimits an essential region between -6.6 and -5.6 kb that together with the 3-kb promoter fragment directs transgene expression in the epaxial myotome of all somites during the appropriate developmental period. These data provide evidence that the partly overlapping expression patterns of Mrf4 and Myf5 in somites are controlled by distinct regulatory elements. We also show that 11.4 kb sequence upstream of MRF4, including the promoter and the somitic control region identified in this study, is not sufficient to elicit target specificity towards the strong Myf5 (-58/-48 kb) enhancer, suggesting that additional yet unidentified elements are necessary to convey promoter selectivity and protect the MRF4 gene from this enhancer.
Using monoclonal untibodics to the tick-borne encephalitis virus (TBE) nonstructural protein NS3 two forms of this protein were rcvc&d in TBE-infcctcd mammalian cells: a full-length rorm (69 kDa) and a short form (49 kDa) which has not been observed before and was tilled NW. Rceombinant plasmids were constructed and various fragmcnu of the TBE NS3 gent were cxpresscd in rabbit rcticulocytc lysate. By analyzing immune precipitates of '5S-labclcd translation products. we could monitor and localize internal clcuvage of NS3, due to which the NS3' protein was gcneratcd.
Several mutationswere introduced into the putative serine protease domain of the tick-borne encephalitis virus NS3 protem and mto a possible internal cleavage site wtthm the protein. The influence of these mutations on proteolytic activity of NS3 protein and NS3' protem formation was tested m vitro. It was found that NS3' formation was not dependent on the activity of the NS3 N-terminal serme protease. Mutations affecting the Ser-138 residue of the NS3 protein prohibited cleavage between NS2B and NS3 protems when the NS2B-NS3 part of the viral genome was expressed m vitro. suggesting the key role of Ser-138 in viral serme protease functioning.
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