Glucocorticoid-dependent transformation by human papillomavirus type 16 E7 coding and 3' noncoding sequences.

The establishment of transformation of primary rodent cells by human papillomavirus (HPV) type 16 DNA requires glucocorticoid hormones (Pater et al., Nature 335, 832-835, 1988). Here we provide evidence by mutational analysis that, in the context of the hormone-regulated HPV 16 promoter/enhancer, the only protein coding sequences of HPV 16 required are those of the E7 gene. Moreover, additional sequences adjacent to the 3' end of E7 coding sequences are also essential for the establishment of the transformed phenotype. Splice donor sites, especially an E7 ORF 3' proximal one, are implicated for this cis-acting function, since specific deletion mutations of these splice sites greatly or completely reduced the frequency of transformation and the level of E7 RNA.     

Structure and chromosomal localization of the human 2',3'-cyclic nucleotide 3'-phosphodiesterase gene

Human brain cDNA clones for the myelin associated enzyme 2' 3' -cyclic nucleotide 3' -phosphodiesterase (CNPase) have been isolated and sequenced. The only 5' untranslated region (UTR) sequence found was that of a human CNPII mRNA, with no direct evidence for a CNPI mRNA. Human CNPase cDNAs were used to isolate genomic clones containing the human CNPase gene which is 9 kb long. Four exons were identified, separated by three introns, and the sequence of each exon and intron/exon boundary has been established.
The polymerase chain reaction (PCR) was used to detect the presence of the human CNPase gene in DNA from a panel of rodent/human somatic cell hybrids. By this means the human CNPase gene was mapped to chromosome 17. In situ hybridization of a human CNPase genomic clone to metaphase chromosomes further localized this gene to chromosomal band 17q21.     

The 5' intergenic, promoter, pseudoexon 3 and complete coding sequences of the hybrid gene KIR2DS3*002

Genomic and mRNA sequences support the KIR2DS3*002 gene being a hybrid of KIR2DS3*00103 and KIR2DS5.     

豚鼠钙调蛋白2基因编码区及3'端非编码区序列分析

目的比较分析豚鼠CaM2基因编码区及3'端非编码区序列,获得丰富的豚鼠CaM2基因生物遗传学信息。方法基因测序获得豚鼠CaM2基因编码区及3'端非编码区序列,通过genebank数据库获得不同种属CaM核苷酸序列,采用DNASTAR软件对CaM2不同部位序列进行多序列的比较分析。结果豚鼠CaM2基因编码区与人、小鼠和大鼠的同源率分别为96.7%、91.4%和90.5%,与人、小鼠、大鼠的CaMl和CaM3基因编码区序列的同源率在80%-85%之间。豚鼠CaM2基因3'端非编码区序列与人、小鼠和大鼠的同源率分别为94.2%、93.3%和90.0%,与人、小鼠、大鼠的CaM3基因3'端非编码区序列的同源率分别为26.8%、29.2%和25.9%,与CaMl的同源率在25%-63%之间。结论豚鼠CaM2基因编码区具有种属和亚型较高同源性特征,而CaM2基因3'端非编码区虽在不同种属中具有很高同源性,但在不同种属与其它亚型比对中同源性较低。     

Homology between coding and noncoding sequences within the human class I HLA antigen gene

In our previous paper, we identified 32 recurring base oligomers (1 decamer, 4 octamers, 9 heptamers and 18 hexamers) within the coding sequence for the mouse class I major histocompatibility (MHC) antigen H-2Kb. The compilation of these recurring base oligomers led to the conclusion that the entire ancestral coding sequence for class 1 MHC antigens evolved from tandem repeats of the one 45 base-long primordial building block base sequence. As with most other mammalian genes, the gene for each class I MHC antigen is an admixture of coding and noncoding segments interspersed with each other. Thus, the status of noncoding segments should be clarified in relation to the concept of the primordial building block. The published 4,122 base-long sequence of human pHLA 12.4 germline gene afforded me an opportunity for clarification. Since the number of recurring base oligomers residing within the entire 4,000 + base-long sequence proved unmanageably numerous, the two portions 1,200 bases in the total length were singled out. Within these portions containing five noncoding and four coding segments, noncoding and coding segments shared 1 nonomer, 3 octamers, 6 heptamers and 5 hexamers; all the above-noted base oligomers were derived from different parts of the same 45 base-long primordial building block. It was thus concluded that not only the coding segments, but the entire ancestral gene for class I MHC antigens evolved from tandem repeats of the 45-base-long primordial building block. This new concept of primordial building block was discussed in relation to the mechanism of evolution by gene duplication.     

Genetic polymorphism in the 3' ntranslated region of human phosphoglucomutase-1

A 317-bp segment of DNA from the 3' region of the human phosphoglucomutase-1 (PGMl) gene has been examined by a non-radioactive technique for the occurrence of single-strand conformation polymorphism (SSCP), Eight phenotypes were detected and attributed to the presence of four alleles. Genetic analysis of 75 unrelated individuals and six CEPH families whose PGMl protein phenotypes were known revealed strong association between the PGMl '+' and '−' isozyme phenotypes and the variation detected in this region, but no association with the PGMl 1 and PGMl 2 isozyme phenotypes. DNA sequence analysis demonstrated the presence of three nucleotide substitutions underlying the alleles, which were located in the untranslated region of the PGMl gene. There was complete correlation between the nucleotide sequence and the phenotype detected by SSCP analysis. This study provides support for the model that the PGMl isozyme polymorphism is determined at two distinct sites in the coding sequence, one coding for the '1' and '2' alleles and the other coding for the '+' and '−' alleles, separated by a region where intragenic recombination occurs.     

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.     

Context of deletions and insertions in human coding sequences

We studied the dependence of the rate of short deletions and insertions on their contexts using the data on mutations within coding exons at 19 human loci that cause mendelian diseases. We confirm that periodic sequences consisting of three to five or more nucleotides are mutagenic. Mutability of sequences with strongly biased nucleotide composition is also elevated, even when mutations within homonucleotide runs longer than three nucleotides are ignored. In contrast, no elevated mutation rates have been detected for imperfect direct or inverted repeats. Among known candidate contexts, the indel context GTAAGT and regions with purine-pyrimidine imbalance between the two DNA strands are mutagenic in our sample, and many others are not mutagenic. Data on mutation hot spots suggest two novel contexts that increase the deletion rate. Comprehensive analysis of mutability of all possible contexts of lengths four, six, and eight indicates a substantially elevated deletion rate within YYYTG and similar sequences, which is one of the two contexts revealed by the hot spots. Possible contexts that increase the insertion rate (AT(A/C)(A/C)GCC and TACCRC) and decrease deletion (TATCGC) or insertion (GCGG) rates have also been identified. Two-thirds of deletions remove a repeat, and over 80% of insertions create a repeat, i.e., they are duplications.     

Structural characterisation of the distal 5' flanking region of the human interleukin-10 gene

Interleukin-10 (IL-10) is an important immunoregulatory cytokine. The recent characterisation of the proximal 5' flanking region of IL-10 led to the identification of the promoter region. Two polymorphic dinucleotide repeats and 10 single nucleotide polymorphisms (SNPs) have been identified and suggested to be useful genetic markers in several diseases. We have sequenced a further 5275 bp from -9296 to -4021 of the distal part of the 5' flanking region of the human IL-10 gene from the cosmid clone pWE15-4/11. Our sequence analysis reveals a high density of Alu-repeats within the IL-10 gene locus, including three novel, related structures which we term Alu-IL10 (A-C). Using three overlapping PCR products spanning 5110 bp of this distal part of the IL-10 gene the following single base pair substitutions were identified: at -8571 C/T, -8531 G/A, -6752 A/T, -6208 G/C, -5402 C/G. In addition a heterozygous three base pair deletion at -7400 was observed. The SNPs at -8571 C/T and -8531 G/A are contained within an Alu-repeat. These data should further the understanding of how the IL-10 gene is controlled in man and how its function may vary between individuals.     

Sequence of the fusion protein gene of human parainfluenza type 2 virus and its 3' intergenic region: lack of small hydrophobic (SH) gene

cDNA clones representing the fusion (F) gene of human parainfluenza virus type 2 (PIV-2) were isolated from cDNA libraries constructed from virus-specific mRNA and genomic RNA, and the complete nucleotide sequence of the F gene was determined. The F gene is 1854 nucleotides long and encodes one long open reading frame of 551 amino acids. The cleavage site for activation of the precursor Fo protein is Thr-Arg-Gln-Lys-Arg. The F gene of PIV-2 is most closely related to those of simian virus 5 (SV5) and mumps virus (MuV). Interestingly, although the HN glycoprotein of PIV-2 shows no relatedness to the HA glycoprotein of measles virus (MV), a distinct homology is found in the F proteins of PIV-2 and MV. As concerns F proteins, paramyxoviruses can be divided into two subgroups; that is, PIV-2, SV5, and MuV belong to one group, and HPIV-1, SV, and PIV-3 belong to the other group. Newcastle disease virus (NDV) and MV are intermediate. Coding regions for small hydrophobic (SH) proteins have been found between the HN and F genes of SV5 and MuV, which are the viruses most closely related to PIV-2. However, such a gene could not be detected in two different strains of PIV-2.     

Chicken IgL variable region gene conversions display pseudogene donor preference and 5' to 3' polarity

Chicken immunoglobulin variable region diversity is generated during B-cell development in the bursa of Fabricius by intrachromosomal gene conversion, resulting in the replacement of sequence blocks within the unique rearranged VL1 and VH1 genes with homologous sequences derived from V region pseudogene segments (psi V). In this report, the nucleotide sequences of 217 gene conversion events in 52 random IgL clones were analyzed to characterize the molecular mechanism of gene conversion. The frequency of psi VL usage as gene conversion donors is shown to depend on the proximity of the psi VL segment to VL1, extent of homology with VL1, and relative orientation of the psi VL segments. Gene conversion events are not observed in the 5' region of homology between psi VL segments and VL1, but are distributed throughout the remainder of the VL1 exon. The 5' ends of individual gene conversion events always begin in regions of homology between the donor psi VL and recipient VL1 gene, whereas the 3' ends can occur in regions of nonhomology and often have nucleotide insertions or deletions. These results suggest a 5' to 3' polarity in the gene conversion mechanism. The implications of our data are discussed in relation to current molecular models of gene conversion.     

Analysis of the in vitro coding properties of the 3' region of turnip yellow mosaic virus genomic RNA

The genomic RNA of turnip yellow mosaic virus (TYMV) codes in vitro for two proteins of 195,000 (195K) and 150,000 (150K) daltons initiated at the same site of the RNA. In the presence of yeast amber suppressor tRNA, synthesis of the 195K protein is reduced in favor of a 210,000-dalton (210K) protein. Using TYMV RNA sequence data and comparison of tryptic peptides of the 150K, 195K, 210K, and coat proteins, we postulate that the silent region between the 195K gene and the coat protein gene located in the 3' region of TYMV genomic RNA is only 14 nucleotides long.     

Enhancement of dengue virus translation: role of the 3' untranslated region and the terminal 3' stem-loop domain

An essential step for a productive infection by the dengue flavivirus (DEN) is translation of the m(7)G-capped, nonpolyadenylated positive-sense RNA genome. We have recently identified sequences within the DEN 3' untranslated region (UTR) that modulate viral translation. Here, we show that the DEN type 2 (DEN2) 3'UTR stimulated translation of m(7)G-capped DEN2 5'UTR-containing reporter mRNAs in baby hamster kidney (BHK) cells compared to a 3' vector sequence. Analogous to the 3' poly(A) tail, the DEN2 3'UTR also enhanced translation of reporter mRNAs containing (i) a nonfunctional A cap, (ii) the 5'UTR of human beta-globin, or (iii) a viral internal ribosome entry site (IRES). In all cases, approximately half of the translation efficiency was due to the terminal 3' stem-loop (3'SL) domain. In addition, the 3'SL domain increased the association of mRNAs with polysomes. Together, these results indicate that the DEN2 3'UTR, mediated in part by the 3'SL domain, enhances translation initiation, possibly after recognition of the 5' cap structure.