Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a procedure that detects the presence of mRNA coding for human beta-myosin heavy chain in small amounts of total, unfractionated RNA isolated from heart or skeletal muscle. The protocol is based on the enzymatic amplification in vitro of a selected 106-bp myosin isotype-specific subregion of this mRNA. The method, which is a modification of the so-called "polymerase chain reaction," requires two synthetic oligonucleotide primers (20-mers), reverse transcriptase, and DNA polymerase I (Klenow fragment). Two principle steps are involved: (i) the selected mRNA subregion is converted into a double-stranded cDNA, and (ii) this cDNA is amplified in 22 synthetic cycles. After gel electrophoresis and blotting the amplification product is identified by hybridization with a third oligonucleotide recognizing the region between the two primer annealing sites, and by restriction mapping. Only mRNA from muscle tissue promoted formation of the amplified 106-bp fragment. We estimate that less than 30,000 beta-myosin heavy-chain mRNA molecules are sufficient to produce a signal. The procedure is fast, specific, and very sensitive. It may be used in muscle gene expression studies with small numbers of cells or even in single muscle fibers.
...
PMID:Enzymatic amplification of myosin heavy-chain mRNA sequences in vitro. 284 Feb 50

Molecular genetic analyses of a 376-kDa Golgi complex (GC) membrane protein (giantin) are described. The immunoglobulin G fraction of a human serum containing antibodies against GC antigens as revealed by indirect immunofluorescence microscopy with Hep-2 cells was used to screen a HeLa cDNA expression library, yielding four overlapping cross-hybridizing clones. Additional cDNA clones were retrieved from a lambda gt11 human thyroid cDNA library or generated by reverse transcriptase-mediated PCR from HeLa cell mRNA. Alignment of the clones resulted in a consensus cDNA of 10,300 bp encoding a protein of 376 kDa. The corresponding mRNA with a size of about 10 kb was detected by Northern (RNA) blotting of HeLa, Hep-G2, and Jurkat cell RNA. Sequence analyses of the protein revealed an extraordinarily high content of heptad repeats with the probability of forming coiled coils similar to the proteins of the myosin family. Five overlapping recombinant proteins covering the entire sequence were synthesized and used for antibody production in rabbits and for affinity purification of human and rabbit antibodies. Indirect immunofluorescence experiments also done with brefeldin A-treated Hep-2 and Pt K1 cells revealed an identical GC staining of both the affinity-purified human and rabbit antibodies. Double labeling experiments with antibodies against the GC marker mannosidase II as well as immunoelectron microscopic studies confirmed the localization of the protein within the GC. A corresponding endogenous large-molecular-mass protein of about 390 kDa was found in [35S]methionine-labeled Hep-2 cell lysates as well as in GC-enriched subcellular fractions from rat liver. The protein as well as the recently described proteins golgin-95 and golgin-160 (M. J. Fritzler, J. C. Hamel, R. L. Ochs, and E. K. L. Chan, J. Exp. Med. 178:49-62, 1993) may belong to a new group of Golgi proteins with a high content of heptad repeats which may exert functions in scaffold formation or vesicle transport. As far as can be concluded from immunological and personally communicated partial cDNA sequence data, the protein seems to be identical with a 400-kDa Golgi protein (giantin) recently described (A. D. Linstedt and H. P. Hauri, Mol. Biol. Cell 4:679-693, 1993). Therefore, we agreed to adopt the name giantin.
...
PMID:Molecular genetic analyses of a 376-kilodalton Golgi complex membrane protein (giantin) 779 69

Brush border myosin-I, or BBMI, constitutes the lateral links that connect in intestinal microvilli the core bundle of actin filaments to the membrane. Although related molecules have been identified in other higher eukaryotic tissues, northern blot analysis has indicated that the distribution of this particular myosin-I isoform is restricted essentially to intestine. Using reverse transcriptase polymerase chain reaction we have identified BBMI in a wide range of tissues including liver and testis. Our results also indicate that in testis the BBMI gene might be alternatively spliced.
...
PMID:Identification of brush border myosin-I in liver and testis. 777 4

In the present study, we have used single chicken blastoderms of defined early developmental stages, beginning with the prestreak stage, stage 1 (V. Hamburger and H. L. Hamilton, J. Morphol. 88:49-92, 1951), to analyze the onset of cardiac myogenesis by monitoring the appearance of selected cardiac muscle tissue-specific gene transcripts and the functional expression of the myocyte enhancer factor 2 (MEF-2) proteins. Using gene-specific oligonucleotide primers in reverse transcriptase PCR assay, we have demonstrated that the cardiac myosin light-chain 2 (MLC2) and alpha-actin gene transcripts appear as early as stage 5, i.e., immediately after the cardiogenic fate assignment at stage 4. Consistent with this observation is the developmental expression pattern of DNA-binding activity of BBF-1, a cardiac muscle-specific member of the MEF-2 protein family, which also begins at stage 5 prior to MEF-2. Differential expression of DNA-binding complexes is also observed with another AT-rich DNA sequence (CArG box) as probe, but the binding pattern with the ubiquitous TATA-binding proteins remains unchanged during the same developmental period. Thus, the cardiogenic commitment and differentiation of the precardiac mesoderm, as exemplified by the appearance of cardiac MEF-2, MLC2, and alpha-actin gene products, occur earlier than previously thought and appear to be closely linked. The onset of skeletal myogenic program follows that of the cardiogenic program with the appearance of skeletal MLC2 at stage 8. We also observed that mRNA for the MEF-2 family of proteins appears as early as stage 2 and that for CMD-1, the chicken counterpart of MyoD, appears at stage 5. The temporal separation of activation of cardiac and skeletal MLC2 genes, which appears immediately after the respective fate assignments, and those of cardiac MEF-2 and CMD-1, which occur before, are consistent with the established appearance of the myogenic programs and with the acquisition pattern of the two tissue-specific morphological characteristics in the early embryo. The preferential appearance of BBF-1 activity in precardiac moesderm, relative to that of MEF-2, indicates that these two protein factors are distinct members of the MEF-2 family and provides a compelling argument in support of the potential role of BBF-1 as a regulator of the cardiogenic cell lineage determination, while cardiac MEF-2 might be involved in maintenance of the cardiac differentiative state.
...
PMID:Differential expression of the myocyte enhancer factor 2 family of transcription factors in development: the cardiac factor BBF-1 is an early marker for cardiogenesis. 803 95

The molecular mechanisms underlying the heterogeneity in contractile properties observed among smooth muscle tissues are unknown. We examined whether part of this diversity might be intrinsic to myosin by comparing structural and enzymatic properties of myosins from two physiologically diverse tissues. Using the reverse transcriptase polymerase chain reaction, we compared avian intestinal smooth muscle and vascular smooth muscle myosin heavy chain (MHC) mRNA. We found that intestinal, but not vascular, MHC mRNA contains an insert of 21 nucleotides, encoding 7 amino acids, in a region near the ATP binding site in the myosin head. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified myosin revealed that the relative mobilities of the previously described intestinal MHC isoforms SM1 (204 kDa) and SM2 (200 kDa) were slower than the corresponding vascular SM1 and SM2 isoforms. Furthermore, antibodies raised against a synthetic peptide corresponding to the deduced amino acid sequence of the intestinal insert strongly recognized intestinal SM1 and SM2 but only weakly recognized the vascular isoforms. The presence of the insert in intestinal myosin correlated with a higher velocity of movement of actin filaments in vitro and a higher actin-activated Mg(2+)-ATPase activity, compared with vascular myosin. Other than the MHC insert, one other structural difference distinguished intestinal and vascular myosins: two isoforms of the 17-kDa myosin light chain were found in vascular myosin, whereas a single isoform was found in intestinal myosin. Exchange of the intestinal myosin light chains onto the vascular MHC did not alter its activity in the in vitro motility assay, suggesting that the 7-amino acid MHC insert is responsible for the different enzymatic activities of vascular and intestinal myosins.
...
PMID:An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. 850 18

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.
...
PMID:An axoplasmic myosin with a calmodulin-like light chain. 865 Feb 20

We demonstrate, using reverse transcriptase-polymerase chain reaction, that, whereas abdominal aorta from rabbit consists almost entirely of myosin heavy chain (MHC) mRNA with no insert at the 5'-terminal coding region, the distributing arteries (femoral and saphenous) begin to show MHC mRNA with the 21-nucleotide insert that encodes seven amino acids in the ATP-binding region located in the myosin head. The femoral/iliac artery contains > 50% inserted mRNA, whereas the more distal saphenous artery contains > 80% inserted mRNA. This insert is also present in the smooth muscle from rat tail artery but is absent in the smooth muscle from rat aorta. The actin-activated ATPase activity of myosin from the rabbit femoral/saphenous artery is 1.7-fold higher than that of the myosin from the aorta. A concomitant increase (about twofold) in the maximum shortening velocity of the saphenous artery, compared with that of the aorta, indicates that the preponderance of the inserted myosin is associated with both an increase in the actin-activated ATPase activity and a larger maximum velocity of shortening. Furthermore, analysis of the 17-kDa essential light chain from the aorta reveals near equal quantities of the 17-kDa light chain isoforms a and b, whereas the myosin from the femoral/ saphenous artery contains predominantly the 17-kDa light chain a isoform. Together, these data indicate that the smooth muscle cells from the small distributing arteries are similar to those of visceral smooth muscle with respect to the expression of myosin isoforms, actin-activated myosin ATPase activity and contractility.
...
PMID:NH2-terminal-inserted myosin II heavy chain is expressed in smooth muscle of small muscular arteries. 917 44

We review the current understanding of the myosin heavy chain (MHC) isoforms and show that the mRNA levels of smooth muscle (SM)1 and SM2 mimic the expressed levels of SM1 and SM2 protein. The reverse transcriptase-polymerase chain reaction technique has been shown to be sufficiently sensitive to examine SM-MHC expression at the single cell level. Most single smooth muscle cells isolated from adult rabbit carotid express both SM1 and SM2. However, expression of these SM-MHC isoforms at the cellular level is nonuniform and highly variable. This work provides a foundation for future investigations as to the possible unique functional characteristics of the SM-MHC isoforms, SM1 and SM2. This methodology may also prove useful when used with mechanical studies to determine the physiological significance of the alternatively spliced myosin isoforms, including the SM-MHC-head and LC17 isoforms.
...
PMID:Myosin isoform heterogeneity in single smooth muscle cells. 918 12

The process of thymic selection is critical for the generation of the mature T-cell repertoire, yet the nature of the self-peptides that serve this function is not known. Several studies suggest that tissue-specific auto-antigens are expressed in the thymus. We initiated this study to examine the expression of a panel of auto-antigens related to several autoimmune diseases in the thymus, peripheral lymphoid organs, and various cell lines. We looked for the expression of these antigens by reverse transcriptase-polymerase chain reaction, fluorescence-activated cell sorter (FACS) analysis, immunoblotting, and immunoprecipitation. We found that in the thymus there is evidence for the expression of a wide variety of disease-related self-antigens including myelin antigens, insulin, cardiac myosin, and retinal S antigen. By FACS analysis, several monoclonal anti-myelin basic protein antibodies were found to bind to immune cells. In Western blotting, we could find in the thymus and other lymphoid organs the expression of myelin basic protein, proteolipid protein, and cyclic nucleotide phosphodiesterase; in contrast, the staining for myelin oligodendrocyte glycoprotein, microtubule-associated Tau protein, and insulin were negative in these organs. The results of these studies confirm that there is evidence for the expression of a variety of auto-antigens in the immune system, both at the mRNA and protein levels, potentially enabling them to participate in the process of thymic education.
...
PMID:Expression of autoimmune disease-related antigens by cells of the immune system. 978 84

Malignant rhabdoid tumor (MRT) is a rare and extremely aggressive malignant tumor in childhood. In this study, an MRT cell line, designated KP-MRT-NS, was established from the ascitic fluid taken from an 11-month-old girl, whose tumor had originated from the left kidney. Ultrastructural findings demonstrated the typical aggregation of whorls of intermediate filaments. Chromosome constitution was described as 46, XX, add (10)(q26)[17]/46, idem, dis (1;2)(q22;q31)[3] based on ISCN (1995) and a del (22)(q11.2) was not found in this cell line. The origin of MRT is controversial, various cellular origins having been proposed because of the phenotypic diversity of MRT. Therefore, in this study, to clarify the origin of MRT, the expressions of cytoplasmic proteins including smooth-muscle-specific proteins (alpha-smooth-muscle actin, basic calponin, smooth-muscle-myosin-heavy-chain isoforms of SM1 and SM2) in the primary-MRT tissue and cell line were analyzed. In the primary-tumor tissue, the expressions of neurofilament, vimentin and alpha-smooth-muscle actin were demonstrated by indirect immunofluorescence. In the KP-MRT-NS cell line, the expression of neurofilament, alpha-smooth-muscle actin, basic calponin and smooth-muscle-myosin heavy chain of SM1 and SM2 isoforms was revealed by immunofluorescence, Western blot and/or reverse transcriptase-polymerase chain reaction (RT-PCR). MyoD1 mRNA, determined as a skeletal-muscle-cell lineage marker, was not expressed in the primary-tumor tissue or in the KP-MRT-NS cell line. According to our findings, the MRT cells are of both neural and smooth-muscle cell phenotypes, and support the neural-crest origin of MRT.
...
PMID:Malignant rhabdoid-tumor cell line showing neural and smooth-muscle-cell phenotypes. 1041 65


1 2 3 4 Next >>

---