Evolutionarily conserved structure of the 3' non-translated region of a Chinese hamster polyubiquitin gene.

From a V79 Chinese hamster genomic library, we isolated a clone containing a polyubiquitin gene (designated as CHUB1), and determined its nucleotide sequence. The coding region of the CHUB1 gene consisted of five direct repeats of the ubiquitin unit with no spacer, followed by a single tyrosine residue. Northern hybridization analysis with a synthesized probe specific to the 3' non-translated region of the CHUB1 gene revealed that it codes for a 1.8 kb mRNA. An evident homology to the human polyubiquitin gene UbB and the chicken UbI gene was observed in the region corresponding to the full extent of the mature mRNA sequence, suggesting that these three genes belong to a common polyubiquitin gene subfamily, and that the sequence in the 3' non-translated region of the CHUB1 gene is unique to this subfamily.     

Characterization and expression of two chicken cDNAs encoding ubiquitin fused to ribosomal proteins of 52 and 80 amino acids

We have determined the complete nucleotide sequence of two chicken cDNAs, Ub-t52 and Ub-t80, encoding ubiquitin fused to ribosomal proteins of 52 and 80 amino acids. The deduced amino acid sequences of the ribosomal proteins are identical or very similar to the homologous human and rat proteins and to the corresponding proteins of other species. Unexpectedly, the ubiquitin moiety of the Ub-t52 protein showed two amino acid substitutions: serine-20 has been replaced by asparagine and serine-57 by alanine. Ubiquitin is a protein strongly conserved during evolution, with no changes in sequence previously reported in vertebrates. Ub-t52 and Ub-t80 are highly expressed in early embryogenesis and during postmitotic stages of spermatogenesis, in parallel with the expression of the polyubiquitin gene UbII. Whereas the 5′ untranslated regions (5′UTRs) of the chicken polyubiquitin mRNAs showed marked differences in mature testes in relation to somatic tissues, no differences were observed in the 5′UTRs of the ubiquitin-ribosomal protein mRNAs. These mRNAs possess a 5′-terminal oligopyrimidine tract that could be used as a mechanism to postpone translation during postmitotic stages of spermatogenesis, as has been proposed in quiescent cells.     

Characterization of a chicken polyubiquitin gene preferentially expressed during spermatogenesis.

We have previously reported that a chicken polyubiquitin gene (Ub II) not expressed under normal or heat shock conditions in chick fibroblasts is transcribed during spermatogenesis [(1987) Nucleic Acids Res. 15, 9604]. The level of Ub II mRNA is several-fold higher in testis cells than in somatic tissues. The gene Ub II possesses characteristic features not seen in the polyubiquitin gene expressed in heat shock conditions (Ub I). The 5' noncoding region of Ub II shows the consensus cAMP regulatory element (CRE) followed immediately downstream by a CA dinucleotide. It has been proposed that this extended CRE may be involved in the coordinate expression of various genes during spermatogenesis.     

Relative levels of methylation in human growth hormone and chorionic somatomammotropin genes in expressing and non-expressing tissues.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.     

Variable methylation of the ribosomal RNA genes of the rat.

Both the pattern and level of rRNA gene methylation vary in the rat. This variation reflects stages in the maturation process and perhaps the level of gene expression in different tissues. We studied methylation at a common site, the inner cytosine of the sequence CCGG, by hybridizing 32P-rRNA to DNA digests obtained with endonuclease Msp I (which cleaves CCGG and CMCGG) and its isochizomer, HpaII (which cleaves only CCGG). In the liver, the changing pattern of rRNA gene methylation reflected the late stages of development: the rRNA genes were mostly unmethylated at 14 days gestation; by 18 days gestation, about 30% of them were methylated, and this level persisted into adulthood. In 18-day DNA, the methylation was uniform, but in adult DNA, the methylation pattern was discontinuous, because otherwise methylated genes contained a demethylated region. Similar developmental changes were observed in brain DNA. In a tissue culture cell line, the change from the continuous to the discontinuous pattern of the methylation could be induced by transformation with Kirsten sarcoma virus. And, in adult tissues, the lowest level of rRNA gene methylation was found in rapidly growing jejunal epithelium, and the highest level, in non-growing spermatozoa.     

巴西橡胶树43 kD橡胶粒子膜蛋白基因的cDNA克隆及表达

对43 kD的橡胶粒子膜蛋白进行了分离纯化和其N端氨基酸序列分析,根据N端氨基酸序列,设计一简并引物,通过3'RACE(Rapid Amplification ofcDNA Ends)的方法,获得了43 kD的橡胶粒子膜蛋白的cDNA.该cDNA含有1 385个核苷酸,含有完整的阅读框架,编码381个氨基酸.在终止密码子下游,包含有一个239bp的3'非编码区.该cDNA由5个首尾相连的重复单元组成,每个单元编码76个氨基酸组成的泛素(ubiquitin)单体.编码43 kD橡胶粒子蛋白的基因具有多个拷贝,在胶乳、叶片和树皮都表达.