水稻逆转录转座子RIRE10拷贝数与LTR区转录活性的鉴定

在水稻第四号染色体的长臂上鉴定了一个结构完整的Ty3型逆转录转座子RIRE10。RIRE10两LTR间的中间区域在gag pol的上游还包含另一个开放阅读框。通过RT PCR与Northern印迹杂交检测到来自LTR区的转录产物 ;根据点杂交结果 ,鉴定出包含中间区域的RIRE10成员的个数以及LTR区的拷贝数。除了 6 5个完整的逆转录转座子所具备的两个LTR外 ,水稻基因组还含有近 90 0个RIRE10的solo LTR。LTR区的转录以及导致solo LTR产生的同源重组可能影响了RIRE10成员在水稻基因组中的转座活性     

The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site

Several retroviruses have recently been shown to promote translation of their gag gene products by internal ribosome entry. In this report, we show that mRNAs containing the human immunodeficiency virus type 1 (HIV-1) gag open reading frame (ORF) exhibit internal ribosome entry site (IRES) activity that can promote translational initiation of Pr55(gag). Remarkably, this IRES activity is driven by sequences within the gag ORF itself and is not dependent on the native gag 5'-untranslated region (UTR). This cap-independent mechanism for Pr55(gag) translation may help explain the high levels of translation of this protein in the face of major RNA structural barriers to scanning ribosomes found in the gag 5' UTR. The gag IRES activity described here also drives translation of a novel 40-kDa Gag isoform through translational initiation at an internal AUG codon found near the amino terminus of the Pr55(gag) capsid domain. Our findings suggest that this low-abundance Gag isoform may be important for wild-type replication of HIV-1 in cultured cells. The activities of the HIV-1 gag IRES may be an important feature of the HIV-1 life cycle and could serve as a novel target for antiretroviral therapeutic strategies.     

Nucleotide sequence of AKV murine leukemia virus.

AKV is an endogenous, ecotropic murine leukemia virus that serves as one of the parents of the recombinant; oncogenic mink cell focus-forming viruses that arise in preleukemic AKR mice. I report the 8,374-nucleotide-long sequence of AKV, as determined from the infectious molecular clone AKR-623. The 5'-leader sequence of AKV extends to nucleotide 639, after which lies a long open reading frame encoding the gag and pol gene products. The reading frame is interrupted by a single amber codon separating the gag and pol genes. The pol gene overlaps the env gene within the 3' region of the AKV genome. The nucleotide sequence of the 5' region of AKV reveals the following features. (i) The 5'-leader sequence lacks any AUG codon to initiate translation of gPr80gag, suggesting that gPr80gag is not required for the replication of AKV. (ii) A short portion of the leader region diverges in sequence from the closely related Moloney murine leukemia virus and appears to be related to a sequence highly repeated in eucaryotic genomes. (iii) As in Moloney murine leukemia virus, there is a potential RNA secondary structure flanking the amber codon that separates the gag and pol genes. This structure might function as a regulatory protein binding site that controls the relative levels of synthesis of the gag and pol precursors. The nucleotide sequence of the 3' region of AKV is compared with sequences reported previously from both infectious and noninfectious molecular clones of AKV.     

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.     

Murine sarcoma virus ts110 RNA transcripts: origin from a single proviral DNA and sequence of the gag-mos junctions in both the precursor and spliced viral RNAs

Our previous studies have argued persuasively that in murine sarcoma virus ts110 (MuSVts110) the gag and mos genes are fused out of frame due to a approximately 1.5-kilobase (kb) deletion of wild-type murine sarcoma virus 349 (MuSV-349) viral information. As a consequence of this deletion, infected cells grown at 39 degrees C appear morphologically normal, producing a 4-kb viral RNA and a truncated gag gene product, P58gag. At 33 degrees C, however, MuSVts110-infected cells appear transformed, producing two viral RNAs, about 4 and 3.5 kb in length, and two viral proteins, P58gag and P85gag-mos. Recent S1 nuclease analyses (Nash et al., J. Virol. 50:478-488, 1984) suggested strongly that at 33 degrees C about 430 bases surrounding the out-of-frame gag-mos junction and bounded by consensus splice donor and acceptor sites are excised from the 4-kb RNA to form the 3.5-kb RNA. As a result of this apparent splicing event, the gag and mos genes seemed to be fused in frame and allowed the translation of P85gag-mos. In the present study, DNA primers hybridizing to the MuSVts110 4- and 3.5-kb RNAs just downstream of the gag-mos junction points were used to sequence these junctions by the primer extension method. We observed that, relative to wild-type MuSV-349 5.2-kb RNA, the MuSVts110 4-kb RNA had suffered a 1,488-base deletion as a result of the fusion of wild-type gag gene nucleotide 2404 to wild-type mos gene nucleotide 3892. This gag-mos junction is out of frame, containing both TAG and TGA termination codons in the reading frame 42 and 50 bases downstream of the gag-mos junction, respectively. Thus, the MuSVts110 4-kb RNA can only be translated into a truncated gag precursor containing an additional C-terminal 14 amino acid residues derived from an alternate mos gene reading frame. Similar analyses of the MuSVts110 3.5-kb RNA showed a further loss of both gag and mos sequences over those deleted in the original 1,488-base deletion. In the MuSVts110 3.5-kb RNA, we found that gag nucleotide 2017 was fused to mos nucleotide 3936 (nucleotide 2449 in the MuSVts110 4-kb genome). This 431-base excised fragment is bounded exactly by in-frame consensus splice donor and acceptor sequences. As a consequence of this splice event, the TAG codon is excised and the restoration of the original mos gene reading frame allows the TGA codon to be bypassed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5'' leader.

The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.