Correlation of HER-2/neu amplification with expression and with other prognostic factors in 1103 breast cancers.
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Breast Neoplasms↗
Biomedical subjects
G J Lee-Chen
Publications and source records attributed to G J Lee-Chen.
Vaccinia virus gene D12L encodes the small subunit of the viral mRNA capping enzyme.
Vaccinia virus gene D12L, which lies between nucleotides 14,350 and 13,487 in the HindIII D fragment, is transcribed at early times in infection and is capable of encoding a protein 287 amino acids in length with a predicted molecular mass of 33,331. A polyclonal antiserum was raised in rabbits to a fusion protein containing 279 amino acids of the D12L protein, and this serum was used to investigate both the time of synthesis and the function of the D12L protein. A combination of Western blot analysis and immunoprecipitation from pulse-labeled and pulse-chased cell extracts demonstrated that the synthesis of a 31-kDa protein begins early in infection, that it reaches a plateau by about 4 hr, and that it is stable in the infected cell. The D12L protein was localized by Western blot analysis of detergent-solubilized virions to the sodium deoxycholate soluble fraction which suggested that it may be a virion core-associated enzyme. Due to the similarity in apparent molecular weight between the D12L protein and the small subunit of the vaccinia mRNA capping complex the anti-D12L antiserum was employed in Western blot analysis of fractions generated during the purification of the virion mRNA capping enzyme. The 31-kDa D12L protein copurified with the virus capping enzyme through chromatography on heparin-agarose and phosphocellulose and also cosedimented with the capping enzyme through a glycerol density gradient. In addition, the anti-D12L antiserum coprecipitated the large subunit of the capping enzyme, confirming that gene D12L encodes the small subunit of the viral mRNA capping enzyme. An insertion mutation which destroys the gene D12L coding sequence was constructed in a plasmid containing a portion of both genes D11L and D12L and this plasmid was used to rescue a ts mutation, in a single step, in the adjacent gene D11L. Southern blot analysis of the re-plaque-purified virus permitted the identification of the mutant virus only when the mutant was propagated in the presence of wild-type helper virus. We concluded from these data that gene D12L is essential for virus propagation in tissue culture.
Base Sequence↗
Transcription and translation mapping of the 13 genes in the vaccinia virus HindIII D fragment.
The vaccinia virus HindIII D fragment is 160,060 bp in length and encodes 13 complete open reading frames [Niles et al. (1986) Virology 153, 96-112; S. L. Weinrich and D. E. Hruby (1986). Nucleic Acids Res. 14, 3003-3016]. We have employed a two-step Northern hybridization protocol using single-stranded DNA probes from M13 recombinants in order to identify the mRNA products from the 13 genes. Six of these genes are expressed only at early times after infection; six are transcribed only at late times; one gene is expressed at both early and late times after virus infection. The D11 gene is transcribed into two late mRNA species, one full-length and the other derived from the 3' one-third of the coding sequence. Translation of hybrid-selected mRNA was carried out in an attempt to identify the protein products encoded by each mRNA. Protein products were found for each early gene but translation was successful for only two of the eight late mRNAs. With the completion of the physical map it is apparent that the early and late genes in the HindIII D fragment are arranged in order to minimize potential interference caused by the expression of closely packed viral genes.
DNA Restriction Enzymes↗
Structure of the transcription initiation and termination sequences of seven early genes in the vaccinia virus HindIII D fragment.
The vaccinia virus HindIII D fragment is 16,060 bp in length and encodes 13 complete genes [E.G. Niles et al. (1986). Virology 153, 96-112; S. L. Weinrich and D. E. Hruby (1986). Nucleic Acids Res. 14, 3003-3016]. Six of these genes are expressed only at early times after infection and one gene is expressed at both early and late times [G. -J. Lee-Chen and E. G. Niles (1988). Virology 163, 52-63]. Transcript mapping by S1 nuclease protection studies was carried out and compared to the results of primer extension analyses, in order to locate map positions of the 5' termini of each early mRNA. The lengths of the products of in vitro transcription, from DNA templates which possess the transcription start regions of each of the early genes, were determined and compared to the lengths of DNA products generated by S1 nuclease protection and primer extension, in order to demonstrate that the 5' termini identified by S1 mapping and primer extension are due to transcription initiation and not to mRNA processing. For each of the early genes in the HindIII D fragment, transcription starts within 25 nucleotides of the translation initiation codon. The precise location of the 3' termini of each early transcript was identified by S1 nuclease mapping. In all but one case, the 3' ends map within 75 nucleotides of the putative transcription termination signal TTTTTNT [G. Rohrmann, L. Yuen, and B. Moss (1986).
Base Sequence↗
Map positions of the 5' ends of eight mRNAs synthesized from the late genes in the vaccinia virus HindIII D fragment.
The map positions of the 5' ends of eight late mRNAs from the vaccinia virus HindIII D fragment were determined by a combination of S1 nuclease-protection studies and by primer extension analysis. For genes D2, D8, D10, and D11, a single set of 5' ends can be observed by S1 nuclease analysis which maps just upstream from the translation start site for each gene. For genes D3, D6, and D11a the situation is more complex. In addition to the ATG proximal family of protected DNA fragments, multiple larger protected DNA fragments map to sites up to several hundred base pairs upstream from the coding region. When primer extension mapping is carried out, large extended products are observed in all cases but that of gene D10. For genes D2, D8, and D13, these large DNA products are heterogenous in length and much longer than the S1 nuclease-protected DNA fragments. This has been observed previously for the late mRNA from the 11K gene by Bertholet et al. [(1987). Cell 50, 153]. In the case of mRNA from genes D3 and D6, however, the lengths of the extended DNA primers agree with the lengths of the S1 nuclease-protected DNA fragments. Therefore, for genes D3 and D6, the 5' regions of the mRNA must be derived from transcription of the DNA sequences upstream from the coding region of each gene. Since the structures at the 5' ends of the late mRNA assume more than one form, there may be multiple pathways for generating late mRNA.
Base Sequence↗
Evolutionarily selected replication origins: functional aspects and structural organization.
A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.
Animals↗
Simian virus 40 DNA replication: functional organization of regulatory elements.
The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G + C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp repeat region 180 or 370 bp upstream of the ori sequence abolishes its enhancing effect, whereas no replication is detected if the 21-bp repeat region is placed downstream of the ori sequence. The dose-dependent enhancement of the 21-bp repeat of SV40 DNA replication as first described in single transfection by Bergsma et al. (D. J. Bergsma, D. M. Olive, S. W. Hartzell, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 79:381-385, 1982) is dramatically amplified in mixed transfection. In the presence of the 21-bp repeat region, the 72-bp repeat region can enhance SV40 DNA replication. In the presence of the 21-bp repeats and a competitive environment, the 72-bp repeat region exhibits a cis-acting inhibitory effect on SV40 DNA replication.
Animals↗