Contribution of polyadenylate sequences to the translational efficiency of globin messenger RNAs.

mRNAs from reticulocyte polysomes were fractionated by chromatography on poly(U)-Sepharose and thermal elution. The molar ratio of alpha- to beta-globin mRNA was found to be 2:1 and 1:1 respectively in short- and long-poly(A) size classes. Translational analyses indicated that the globin mRNAs containing long poly(A) tracts (with a mean length of about 70 nucleotides) directed protein synthesis with higher rates than did mRNA containing short poly(A) tracts (15-35 nucleotides). Experiments performed with sub-saturating mRNA concentrations showed that the digestion with RNAase H induced a decrease in the translational capacity of both globin mRNAs and an increase in the alpha- to beta-globin synthesis ratio. No correlation was observed between the size of the poly(A) tail in mRNA and the optimal K+ requirement for translation.     

Methylated nucleotides block 5' terminus of HeLa cell messenger RNA.

Polyadenylylated [poly(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poly(A)+ RNA was calculated from the 3H/14C ratios and known degrees of methylation of 18S and 28S ribosomal RNAs. All four 2'-O-methylribonucleosides, but only two base-methylated derivatives, 7-methylguanosine (7MeG) and 6-methyladenosine (6MeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyl-labeled ribonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5' terminus of the poly(A)+ RNA and could be removed by periodate oxidation and beta elimination. Evidence for a 5' to 5' linkage of 7MeG to adjacent 2'-O-methylribonucleosides through at least two and probably three phosphates to give structures of the type 7MeG5'ppp5pNMep- and 7MeG5'ppp5'NMepNmep- was presented. The previous finding of similar sequences of methylated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5' termini may be a characteristic feature of mRNAs that function in eucaryotic cells.     

不同培养体系对鼠-猪异种嵌合胚胎发育和嵌合能力的比较

近年来,随着干细胞分化与再生医学研究的不断深入,异种嵌合已成为当前干细胞和再生医学领域的热点问题,并有望为未来解决器官移植供体来源严重短缺等再生医学难题开辟新的方向。异种嵌合以及异种器官再造过程中面临众多科学问题和技术难题,而异种嵌合过程中嵌合胚胎时期的选择,后续培养液的选择以及这些环节所造成的供体细胞与受体胚胎之间的发育平衡成为建立异种器官再造的第一个科学问题。猪由于具有与人类器官大小相似、繁殖快等特点,成为异种嵌合最适合的潜在研究对象。为了提高鼠-猪异种嵌合胚胎中小鼠供体细胞——诱导多潜能干细胞(Induced pluripotent stem cells,i PSCs)的存活率和增殖率,我们尝试以i PSCs培养液(N2B27)以及N2B27→PZM-3梯度更换的培养液(N2B27(3.5 h))作为研究异种嵌合胚胎体外发育培养的对象,并与猪胚胎培养液(PZM-3,Porcine zygotic medium)体系下发育进行比较,从而评价了这3种培养液在8-细胞和囊胚期注射后,对嵌合胚胎后续发育的影响及嵌合情况。结果显示,8-细胞期注射后,PZM-3不仅对嵌合胚胎的后续发育较为有利,更有利于小鼠i PS嵌合到猪胚胎中;囊胚期注射后3种培养体系下GFP阳性嵌合率差异不显著,但其嵌合率显著低于8-细胞期嵌合率。结果表明,PZM-3培养体系更有利于鼠-猪异种嵌合胚胎的体外发育,对8-细胞期胚胎进行嵌合操作有益于提高鼠-猪异种嵌合后胚胎的嵌合率。     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.     

In vitro translation of the three bacteriophage phi 6 RNAs.

In vitro translation of the three single-stranded RNAs transcribed in vitro by bacteriophage phi 6 RNA polymerase revealed that the large RNA codes for phage proteins P1, P2, P4, and P7, the medium RNA codes for P3, P6, and P10, and the smaller RNA for P5, P8, and P9.     

Translation of RNAs Synthesized In Vivo and In Vitro from Bacteriophage SP82 DNA

The synthesis of 69 phage-specific polypeptides during the infection of Bacillus subtilis with bacteriophage SP82 was detected by pulse-labeling, one-dimensional electrophoresis, and autoradiography. SP82 virions were found to contain approximately 22 polypeptides, most of which were synthesized late in infection; evidence was obtained for the processing of the major virion protein. RNAs extracted at different times during infection were translated by using an Escherichia coli cell-free extract. Only smaller-molecular-weight peptides were produced efficiently in vitro; in the 9,000- to 60,000-molecular-weight range, 50 to 60% of the peptides synthesized in vivo were produced by translation of RNAs extracted from infected cells. Eight of the virion peptides were produced by in vitro translation of RNAs extracted from infected cells. RNAs were synthesized under defined conditions by RNA polymerase extracted from uninfected B. subtilis and by polymerases isolated from cells 8 and 20 min after infection with SP82. Translation of these RNAs yielded characteristic and different patterns of polypeptides. Nine of the 12 polypeptides produced by translation of RNAs synthesized by the host polymerase corresponded in mobility to peptides appearing in vivo in the 0 to 3 and 3 to 6 min intervals of pulse-labeling after infection; 12 of the 25 peptides synthesized from RNAs produced by polymerase extracted 8 min after infection corresponded in mobility to peptides detected in vivo 8 min after infection, and 15 of the 22 peptides directed by RNAs made by the polymerase isolated 20 min after infection corresponded to peptides present in vivo late in infection. Five of the peptides produced in vitro from the latter RNA corresponded to virion peptides.