Marschalek R, Amon-Böhm E, Stoerker J, Klages S, Fleckenstein B, Dingermann T
Institute für Biochemie, Medizinischen Fakultät, Universität Erlangen-Nürnberg, FRG.
Nucleic Acids Res. 1989 Jan 25;17(2):631-43. doi: 10.1093/nar/17.2.631.
Through computer analysis of a human cytomegalovirus (HCMV) genomic region, previously identified to be homologous to human genomic DNA, an element showing significant similarity to the 3'-internal control region (3'-ICR or B-block) of a eukaryotic RNA polymerase III promoter could be detected. This region-located on the EcoRI b fragment within the UL segment of the viral genome of HCMV strain AD 169-cannot be transcribed in vitro in an RNA polymerase III specific transcription system. However, this part of the viral genome is able to compete for components of the RNA polymerase III transcription complex as shown in template exclusion experiments and by gel retardation assays. Two different synthetic oligonucleotides complementary to the 3'-ICR and to nucleotides located immediately downstream of this promoter element can anneal specifically to a HCMV-encoded ribonucleic acid (termed CMER) synthesized in human foreskin fibroblasts (HFF) late in virus replication. As a consequence of identifying the transcription initiation point by primer extension analyses the position of the 5'-internal control region (5'-ICR or A-block) of the CMER gene could be uncovered. Both identified control regions (the A-block as well as the B-block) of the transcription unit exhibit significant similarities to corresponding regulatory elements of other class III genes, including virus encoded class III genes. Initiation of in vivo transcription occurs 15 nucleotides upstream of the 5'-border of the 5'-ICR and the two non-contiguous gene internal promoter elements are separated by 79 nucleotides.
通过对人巨细胞病毒(HCMV)基因组区域进行计算机分析,该区域先前被确定与人类基因组DNA同源,结果发现一个与真核RNA聚合酶III启动子的3'-内部控制区(3'-ICR或B区)具有显著相似性的元件。该区域位于HCMV AD 169株病毒基因组UL片段内的EcoRI b片段上,在RNA聚合酶III特异性转录系统中无法在体外转录。然而,如模板排除实验和凝胶阻滞分析所示,病毒基因组的这一部分能够竞争RNA聚合酶III转录复合物的成分。两种与3'-ICR及该启动子元件紧邻下游核苷酸互补的不同合成寡核苷酸,能够特异性退火至在人包皮成纤维细胞(HFF)病毒复制后期合成的一种HCMV编码的核糖核酸(称为CMER)。通过引物延伸分析确定转录起始点后,CMER基因5'-内部控制区(5'-ICR或A区)的位置得以揭示。转录单位的两个已确定的控制区(A区和B区)与其他III类基因(包括病毒编码的III类基因)的相应调控元件具有显著相似性。体内转录起始于5'-ICR 5'边界上游15个核苷酸处,两个不连续的基因内部启动子元件相隔79个核苷酸。