Analysis of the size distribution of first-strand cDNA molecules.

We have developed a method to analyze the size distribution of the first-strand cDNA molecules corresponding to given mRNA species. First-strand molecules synthesized from cytoplasmic polyadenylated RNAs are separated by electrophoresis on an alkaline agarose gel, and a Southern blot hybridization is performed. As an example, we analyzed the first-strand molecules corresponding to the human c-myc mRNAs. This method can be used to determine whether full-length, first-strand molecules corresponding to an mRNA species to be cloned are synthesized efficiently. Interestingly, this method allows one to analyze full-length, first-strand cDNA molecules with a much higher resolution than Northern blot analysis of mRNA molecules. This method can therefore be used to discriminate between the multiple mRNA species transcribed from a given gene or the homologous mRNA species transcribed from a given gene family.

Characterization of new human c-myc mRNA species produced by alternative splicing.

Characterization of two human c-myc cDNAs corresponding to the mRNAs 2.5 and 3.1 kb in length transcribed from P0 previously demonstrated the existence of alternative acceptor sites at the end of intron 1 and intron 2, respectively [Bentley, D.L. and Groudine, M. (1986) Mol. Cell. Biol. 6, 3481-3489]. We investigated the use of these alternative acceptor sites in each c-myc mRNA species. We characterized cDNAs corresponding to c-myc mRNAs transcribed in the SW613-S human carcinoma cell line. The use of the alternative acceptor site at the end of intron 1 was demonstrated in two out of 10 cDNAs corresponding to the 3.1-kb mRNA transcribed from P0 and in three out of 10 cDNAs corresponding to the mRNAs transcribed from P1 or P2. The use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from P0. The mRNAs resulting from the use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from P0. The mRNAs resulting from the use of this acceptor site would encode for a variant form of the p67 polypeptide lacking one amino-acid residue. Conversely, the use of the alternative acceptor site at the end of intron 2 was not found in any of the cDNAs corresponding to the mRNAs transcribed from P0 (0/10), from P1 or P2 (0/10) and from P3 (0/10). In the course of this study, we isolated a cDNA corresponding to another new c-myc mRNA species. This mRNA is produced by alternative splicing within intron 1 and encodes only for p64.

A human homolog of the S. cerevisiae HIR1 and HIR2 transcriptional repressors cloned from the DiGeorge syndrome critical region.

The DiGeorge syndrome (DGS) is a developmental disorder affecting derivatives of the third and fourth pharyngeal pouches. DGS patients present an interstitial deletion in one of their two chromosomes 22. Cosmid DAC30 was mapped to the DGS smallest critical region. Iterative cDNA library screening initiated with a DAC30 gene fragment candidate yielded a cDNA contig whose assembled nucleotide sequence is consistent with the widely transcribed, 4.2-4.4 kb long, messengers detected by northern analysis. The deduced protein sequence, 1017 amino acids in length, entirely encompasses the 766 amino acids previously designated as TUPLE1. The completed protein has been renamed HIRA because it contains various features matching those found in HIR1 and HIR2, two repressors of histone gene transcription characterized in the yeast Saccharomyces cerevisiae. Strikingly alike in their N-terminal third, HIRA and HIR1 contain seven copies of the WD repeat, a motif implicated in protein-protein interactions, suggesting that they might define a new subfamily of functionally homologous proteins. The remainder of the human polypeptide highly resembles a corresponding fragment in HIR2. We propose that HIRA, alone, could have a part in mechanisms of transcriptional regulation similar to that played by HIR1 and HIR2 together. The presence of a single copy of the HIRA gene in DGS patients possibly accounts for some of the abnormalities associated with this syndrome.

Efficient second-strand cDNA synthesis using T7 DNA polymerase.

We present a second-strand cDNA synthesis method that takes advantage of both the very high processivity and the very high 3' exonuclease activity of T7 DNA polymerase. The first strand is synthesized with reverse transcriptase using oligo(dT) as a primer. After alkaline hydrolysis of the mRNA template, a tract of dT residues is synthesized with terminal transferase at the 3' end of the first strand. The second strand is synthesized using oligo(dA) as a primer. Several oligo(dA) molecules probably anneal to the poly(dT) tract. Because the 3' exonuclease activity of T7 DNA polymerase is very high, the region of the tract annealed to these oligo(dA) molecules is digested. However, the region of the tract annealed to the very oligo(dA) molecule used as a primer for second-strand synthesis is protected. The resulting cDNA molecules could be cloned with a high efficiency. The size distribution of cloned c-myc DNAs was estimated by Southern blot analysis of phage DNA prepared from the amplified library and by analysis of isolated clones. The results indicate that this method allows to obtain full-length cDNA clones with a high efficiency.

Cloning and sequence analysis of the beta subunit of the human translocon-associated protein.

A cDNA corresponding to the beta subunit of the human translocon-associated protein was cloned and sequenced. The polypeptide is 183 amino acids long and 96% homologous to its canine counterpart. Both polypeptides contain a cleavable signal sequence, an NH2-terminal domain extruding in the endoplasmic reticulum lumen, a transmembrane domain and a COOH-terminal domain located in the cytoplasm.

Tumorigenicity of the met proto-oncogene and the gene for hepatocyte growth factor.

The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). It was previously shown that, like the oncogenic tpr-met, the mouse met proto-oncogene transforms NIH 3T3 cells. We have established NIH 3T3 cells stably expressing both human (Methu) and mouse (Metmu) met proto-oncogene products. The protein products are properly processed and appear on the cell surface. NIH 3T3 cells express endogenous mouse HGF/SF mRNA, suggesting an autocrine activation mechanism for transformation by Metmu. However, the tumor-forming activity of Methu in NIH 3T3 cells is very low compared with that of Metmu, but efficient tumorigenesis occurs when Methu and HGF/SFhu are coexpressed. These results are consistent with an autocrine transformation mechanism and suggest further that the endogenous murine factor inefficiently activates the tumorigenic potential of Methu. The tumorigenicity observed with reciprocal chimeric human and mouse receptors that exchange external ligand-binding domains supports this conclusion. We also show that HGF/SFhu expressed in NIH 3T3 cells produces tumors in nude mice.

Structure, tissue-specific expression, and transforming activity of the mouse met protooncogene.

A 6.7-kilobase met complementary DNA (cDNA) was isolated from a pcD cDNA library prepared from C3H mouse fibroblast cell line polyadenylated RNA. Sequence analysis of 6.7-kilobase met cDNA insert revealed that it contained the entire open reading frame and shared an overall homology of 88.1% with the human met gene. Using the mouse met cDNA as probe, high levels of met expression were observed in the kidney, brain, lung, skin, and embryonic tissue as well as in several factor responsive mouse myeloid cell lines. Under SV40 promoter control, the mouse met protooncogene cDNA in the pCD vector was able to transform NIH 3T3 cells. These transformed cells possess multiple copies of mouse met cDNA and exhibit properties of malignant cells, including growth in soft agar and induction of tumors in nude mice. Tumor explant cell lines analyzed by Western blot also reveal the presence of high levels of Mr 170,000 and 140,000 met protein product(s).

A promoter for the highly spliced EBNA family of RNAs of Epstein-Barr virus.

A transcription start for the highly spliced EBNA group of RNAs in B95-8 cells has been identified in the short unique region of the virus genome. This promoter is used in many (but not all) human cell lines carrying Epstein-Barr virus, including a tightly latent human lymphoblastoid cell line. Another promoter for the EBNA RNAs was described previously in the internal repeat region of the virus genome. The existence of these alternative promoters may be important for differential control of EBNA gene expression.

Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells.

The pattern of Epstein-Barr virus (EBV) RNAs expressed in Raji cells superinfected with P3HR1 EBV was examined. RNAs whose expression was of an immediate-early type (resistant to treatment of the cells with anisomycin) were identified. These RNAs, encoding the EBV reading frames BZLF1 and BRLF1, were probably expressed from defective virus within the P3HR1 preparation, and some of them were responsible for the induction of the EBV productive cycle in the Raji cells. The structures of the B95-8 RNAs equivalent to the anisomycin-resistant RNAs were determined. The RNA encoding the BZLF1 reading frame contained two splices which extended and modified the reading frame from that previously described.

Mapping of the gene coding for Epstein-Barr virus-determined nuclear antigen EBNA3 and its transient overexpression in a human cell line by using an adenovirus expression vector.

The open reading frame which lies within the Epstein-Barr virus (EBV) T2 cDNA isolated by Bodescot et al. (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:2611-2620, 1986) was inserted into a eucaryotic expression vector containing a strong adenovirus promoter. The T2 cDNA contains viral genomic sequences from the short BLRF3 open reading frame fused to the adjacent BERF1 long open reading frame. After transfection of human cells, the recombinant plasmid directed the expression of a 140-kilodalton protein. The expressed protein had the same molecular weight, subcellular localization, and immunological characteristics as the EBV-determined nuclear antigen EBNA3, which is made in lymphocytes latently infected with EBV. Immunoprecipitation of extracts of transfected cells labeled with [32P]phosphoric acid showed that the EBNA3 protein is phosphorylated.

Epstein-Barr virus mRNAs produced by alternative splicing.

The structure of Epstein-Barr virus mRNAs transcribed in B95-8 cells has been studied by cDNA cloning and sequencing. We present here the analysis of four cDNAs. The corresponding mRNAs are probably transcribed from a single promoter located in the US region. They are produced by alternative splicing of exons transcribed from the US, IR and UL regions. The exons are spread over 100 kbp. The exons from the IR region constitute a unit which is repeated several times. The cDNAs share the exons from the US and IR regions. Some of the cDNAs also share some of the exons from the UL region. Each cDNA contains a long open reading frame or the 5' end of a long open reading frame which ends several hundred nucleotides downstream on the viral genome. The 5' untranslated regions are unusually long. Three mRNA species differing in their 5' untranslated regions may encode for the nuclear antigen EBNA-1. The other mRNAs encode for polypeptides which may not have any common region.

An Epstein-Barr virus transcription unit is at least 84 kilobases long.

We have studied the structure of the Epstein-Barr virus mRNAs expressed in B95-8, a productively-infected Marmoset cell line established from in vitro-infected B-lymphocytes. We constructed a cDNA library from the cytoplasmic polyadenylated RNAs of B95-8 in the lambda gt10 bacteriophage. We present here the analysis of a 3.5 kbp cDNA containing exons transcribed from the US, IR and UL regions of the viral genome. The corresponding transcription unit is at least 84 kbp long. Two exons are transcribed from the US region, five from the IR region and two from the UL region. The exons from the IR region consist of two tandem repeats of a unit containing two exons, 66 and 132 nucleotides, and of a third copy of the 66 nucleotide exon. The exons from the UL region contain an open reading frame coding for a 944 amino acid polypeptide. The C-terminal end of this polypeptide harbors three types of repeated sequences. The corresponding mRNA is the second described of a family of mRNAs produced by alternative splicing of exons transcribed from the US, IR and UL regions.

Spliced RNA from the IR1-U2 region of Epstein-Barr virus: presence of an open reading frame for a repetitive polypeptide.

We have constructed a cDNA library from the cytoplasmic RNAs of Raji cells, a Burkitt's lymphoma cell line latently infected with Epstein-Barr virus. We report here the characterization of a cDNA representing a spliced RNA transcribed from the IR1-U2 region of the viral genome. The cDNA is 1007 bp long. The 5' region contains three tandem repeats of two exons, 66 and 132 bp, which are transcribed from the IR1 repeats. The 3' region is formed from four exons transcribed from U2. An open reading frame extends from the 5' end to position 784, and includes the repeats. This reading frame presumably corresponds to the carboxy-terminal 261 amino acids of a polypeptide containing several repeats of a 66 amino acid sequence. Since it would be encoded by the IR1-U2 region of the viral genome, the putative polypeptide might be involved in the process of growth-transformation of B-lymphocytes.