Structural organization and nucleotide sequence of mouse c-myb oncogene: activation in ABPL tumors is due to viral integration in an intron which results in the deletion of the 5'' coding sequences.

Bacteriophage libraries of mouse DNA were screened for sequences homologous to the v-myb oncogene and two overlapping clones containing the v-myb related region were isolated. Restriction enzyme mapping, heteroduplex analysis and nucleotide sequence analysis revealed the presence of nine exons. Six of these exons are homologous to the v-myb region while the other three exons are derived from the 5' region which is deleted in the viral oncogene. The sequences downstream to the sixth v-myb exon are not included in the 17 kbp of DNA sequences analyzed in this study. Comparison of the structure of the normal c-myb clone with its rearranged couterpart present in plasmacytoid lymphosarcomas revealed that the rearrangements occur in this locus as a result of viral integration. Present studies demonstrate that such a viral insertion interrupts the c-myb coding region at a region identical to that observed in the generation of the v-myb gene of avian myeloblastosis virus and results in the synthesis of mRNAs that lack the same 5' coding region.     

Transduction of c-myb into avian myeloblastosis virus: locating points of recombination within the cellular gene.

The oncogene (v-myb) of avian myeloblastosis virus apparently arose by transduction of nucleotide sequences from a cellular gene (c-myb). In c-myb the nucleotide sequences that formed v-myb exist at seven distinct regions separated by nontransduced stretches of sequence that are flanked by eucaryotic splice signals. By contrast, the sequences at the outside boundaries of the transduced region of c-myb do not resemble splice sites. We mapped the nucleotide sequences that are homologous to the ends of v-myb with respect to the exons and introns of c-myb. The results indicate that the leftward recombination between c-myb and the transducing retrovirus occurred within an intron of the cellular gene, whereas the rightward recombination took place in an exon of c-myb. Transduction of c-myb sequences may therefore have involved a DNA rearrangement.     

Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.     

Isolation and characterization of the human apolipoprotein A-II gene. Electron microscopic analysis of RNA:DNA hybrids, nucleotide sequence, identification of a polymorphic MspI site, and general structural organization of apolipoprotein genes

Three cloned apolipoprotein A-II genes were isolated from a human genomic cosmid library constructed in our laboratory. An approximately 3-kilobase HindIII insert containing the entire gene was analyzed by RNA:DNA hybridization and electron microscopy. The apo-A-II gene was found to consist of 4 exons and 3 intervening sequences (IVS), and the lengths of each exon and IVS were estimated by direct observation of the hybrids. The entire approximately 3-kilobase HindIII insert was sequenced. The 5' end of the gene was determined by primer extension. The DNA sequence confirms the presence of 4 exons and 3 IVS: exon 1, 34 nucleotides; exon 2, 76 nucleotides; exon 3, 133 nucleotides; exon 4, 230 nucleotides; IVS-I, 169 nucleotides; IVS-II, 299 nucleotides; and IVS-III, 396 nucleotides. A "TATA box" is located at position -29 from the CAP site. A "CAT box" is present at position -78. A "TG" element consisting of (TG)19 is identified at the 3' end of IVS-III. Furthermore, an enhancer core sequence, CTTTCCA, is identified at position -355 in the 5' flanking sequence. At positions -497 to -471 upstream from the CAP site is a stretch of 27 nucleotides that show high homology to stretches of 5' flanking sequences in the apo-C-II, apo-A-I, apo-E, and apo-C-III genes. An Alu dimer sequence is located approximately 300 nucleotides from the 3' end of the gene. Within this Alu sequence, we have identified a polymorphic MspI site. Restriction fragment length polymorphism involving this site has been previously shown to correlate with apo-A-II levels and high density lipoprotein structure. Analysis of conformation by Chou-Fasman analysis and by the helical hydrophobic moment of Eisenberg et al. (Eisenberg, D., Weiss, R. M., and Tergwillager, T. C. (1982). Nature (Lond.) 299, 371-374) indicates that in all of the 5 apolipoproteins characterized at the nucleotide level to date, i.e. apo-C-II, apo-A-II, apo-E, apo-A-I, and apo-C-III, the 2 IVS within the peptide coding regions of the gene tend to occur at regions corresponding to the surface of the polypeptide chain and divide the protein into distinct functional domains.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Topography of the three late mRNA''s of polyoma virus which encode the virion proteins.

The three cytoplasmic polyadenylated mRNA's which separately encode the three capsid proteins (VP1, VP2, and VP3) of polyoma virus were mapped on the viral genome by one- and two-dimensional gel electrophoreses of nuclease S1-resistant RNA-DNA hybrids. The mRNA's, which we designated mVP1, mVP2, and mVP3 to indicate the coding functions deduced from the cosedimentation of the RNAs and the messenger activities, comprise an overlapping set of 3'-coterminal molecules which also share a heterogeneous family of noncoding 5'-terminal regions (Flavell et al., Cell 16:357--371, 1979; Legon et al., Cell 16:373--388, 1979). The three species differ in the length of the 3' colinear coding region which is spliced to the 5' leader sequences. The common polyadenylated 3' end maps at map unit 25.3. The 5' ends of the colinear bodies of mVP1, mVP3, and mVP2 map at 48.5, 59.5, and 66.5 map units, respectively. An examination of the polyoma virus DNA sequence (Arrand et al., J. Virol. 33:606--618, 1980) in the vicinities of splicing sites approximated by the S1 gel mapping data for sequences common to the ends of known intervening sequences allowed prediction of the precise splice points in polyoma virus late mRNA's. In all three cases, the leader sequences are joined to the mRNA bodies at least 48 nucleotides before the translational initiation codon used in each particular messenger. The start signal which functions in each mRNA is the first AUG (or GUG) triplet after the splice junction.     

Structure of the protein encoded by the chicken proto-oncogene c-myb.

The retroviral oncogene v-myb arose by transduction of the chicken proto-oncogene c-myb. We isolated and sequenced cDNA that represents the entire coding domain of chicken c-myb. By transcribing the cDNA into mRNA in vitro and then translating the RNA, we were able to document the integrity of the cDNA and to identify the codon responsible for initiation of translation from c-myb. Two different alleles of v-myb are extant, one in the genome of avian myeloblastosis virus (AMV) and the other in the genome of erythroblastosis virus 26 (E26V). The proteins encoded by the AMV and E26V alleles of v-myb differ from the product of c-myb in three ways: at their amino termini, they lack 71 and 80 amino acids respectively; at their carboxy termini, they are deficient in 199 and 278 residues; and 11 substitutions of amino acids are scattered throughout the product of AMV allele, whereas the product of the E26V allele contains only a single substitution. The structural origins of tumorigenicity by v-myb and the biological functions of c-myb remain enigmatic. The findings and molecular clones described here should now permit a systematic exploration of these enigmas.     

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5''-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.     

Chromosomal reallocation of the chicken c-myb locus and organization of 3'-proximal coding exons

In the course of our studies concerning the tissue-specific expression of the c-myb proto-oncogene, we have established the nucleotide sequence of the chicken c-myb 3'-proximal coding exons. In situ hybridization performed with different genomic DNA probes corresponding to nearly all the c-myb gene allowed us to localize the corresponding locus on the large acrocentric chromosome 3 in chicken. Our sequencing data also indicate that the 3'-proximal noncoding sequences represented in c-myb mRNA species are derived from non-contiguous exons.     

Cloning and characterization of a third type of human alpha-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes.     

Multiple abnormal beta-hexosaminidase alpha chain mRNAs in a compound-heterozygous Ashkenazi Jewish patient with Tay-Sachs disease

Abnormal beta-hexosaminidase alpha chain cDNA clones were isolated from fibroblasts of an Ashkenazi Jewish patient with Tay-Sachs disease. Four abnormal cDNA clones were sequenced in their entirety. We showed previously that three of these mRNAs retained intron 12 with a mutation from G to C at the 5' donor site and that the patient was heterozygous with respect to this splicing defect (Ohno, K., and Suzuki, K., (1988) Biochem. Biophys. Res. Commun. 153, 463-469). One clone retained, in addition to intron 12, intron 13, which was truncated and polyadenylated due to a polyadenylation signal within intron 13. The fourth clone did not contain intron 12 and was missing exon 12. Some of these abnormal mRNAs were also missing one or more of upstream exons. The regions of exon 12-intron 12 and of upstream exons were evaluated in a total of 30 clones, including those completely sequenced, by restriction mapping and Southern analysis with appropriate probes. Of the 25 cDNA clones that included the exon 12-intron 12 region, 11 contained the exon 12-intron 12 sequence with the junctional transversion, and 11 were missing both exon 12 and intron 12. Among the 12 clones that included the region of exon 3-exon 9, 7 were missing one or more of upstream exons. Three clones gave results expected of normal cDNA in the region of exons 12 and 13. One of the three, furthermore, was 3.6-kilobases long and contained the completely normal beta-hexosaminidase alpha chain mRNA sequence on the 3' side and an abnormal 1.7-kilobase segment at the 5' end. These findings suggest that the splicing defect results in either retention of intron 12 or skipping of exon 12 in approximately equal proportions and that remote upstream exons are also frequently excised out. The three clones that were normal in the exon 12-intron 12 region could have derived from the other yet-to-be-characterized mutant allele. However, we were unable to obtain firm evidence that the abnormal upstream sequence is directly related to Tay-Sachs disease.     

Generation of altered transcripts by retroviral insertion within the c-myb gene in two murine monocytic leukemias

Two murine monocytic leukemia cell lines, WEHI-265 and WEHI-274, were found to carry a rearranged c-myb gene. The rearrangements are due to insertion of a deleted Moloney murine leukemia virus (Mo-MLV) provirus in the 5' region of the c-myb gene and thus are similar to rearrangements in the ABPL tumors (G. L. C. Shen-Ong, M. Potter, J. F. Mushinski, S. Lavu, and E. P. Reddy, Science 226:1077-1080, 1984). In each cell line, the retroviral insertion has induced high levels of two aberrant RNA species, which, as in the ABPL tumors (G. L. C. Shen-Ong, H. C. Morse, M. Potter, and J. F. Mushinski, Mol. Cell. Biol. 6:380-392, 1986), contain both viral (Mo-MLV) and cellular (myb) sequences. Both species lack the sequences encoding the amino terminus of the c-myb protein and thus could encode a protein which, like the v-myb gene products (and the predicted ABPL myb proteins), is truncated at the amino terminus. We have found that the larger (5.3 kilobase [kb]) and more abundant of the tumor-specific myb RNAs was predominantly nuclear, while the smaller species (3.9 kb) was cytoplasmic. Furthermore, our data imply that the 3.9-kb RNA was derived from the 5.3-kb RNA by an additional splice which utilized a cryptic splice acceptor site within the viral gag sequences. On the basis of subcellular distribution and predicted translational potential, we conclude that the 3.9-kb RNA is probably the mRNA which encodes a truncated myb protein. We also show that, due to different insertion points in W265 and W274, the W274 myb RNAs contained sequences from a c-myb exon upstream of the exons represented in the W265 (and ABPL) RNAs. The significance of our findings with regard to transformation by myb in these tumors is discussed.     

Rat liver NAD(P)H:quinone reductase: isolation of a quinone reductase structural gene and prediction of the NH2 terminal sequence of the protein by double-stranded sequencing of exons 1 and 2

Recently two reports [J. A. Robertson et al. (1986) J. Biol. Chem. 261, 15794-15799 and R. M. Bayney et al. (1987) J. Biol. Chem. 262, 572-575] have appeared concerning the nucleotide sequence of quinone reductase cDNA clones. Although the cDNA clones are virtually identical, they diverge in the 5' region that encodes the NH2 terminus of the protein. In order to clarify the sequence of this region, we have isolated quinone reductase clones from a rat genomic library using a cDNA clone, pDTD55, isolated and characterized by our laboratory. We have determined the sequence of exons 1 and 2 of the structural gene by double-stranded sequencing using oligonucleotide primers. The sequence of exons 1 and 2 of the quinone reductase structural gene along with our previous nucleotide sequence analysis of pDTD55 as well as conventional amino acid sequence analysis of the purified protein indicates that quinone reductase is composed of 274 amino acids with a molecular weight of 30,946. These data agree with the published sequence of lambda NMOR1 reported by Robertson et al.     

Cloning and nucleotide sequence analysis of the dog insulin gene. Coded amino acid sequence of canine preproinsulin predicts an additional C-peptide fragment

A 4.0-kilobase HindIII/EcoRI-cleaved dog genomic DNA fragment was shown to contain the dog insulin gene by restriction mapping using a human insulin cDNA probe. This fragment was subsequently cloned in a lambda vector, and the nucleotide sequence of the dog insulin gene was determined. As in several other species, the insulin gene of the dog is interrupted by two intervening sequences, one of 151 base pairs located in the 5' untranslated region and the other of 264 base pairs occurring within the codon of the 7th amino acid of the C-peptide. Translation of the nucleotide sequence in one frame revealed the primary structure of canine preproinsulin. An interesting feature of the coded amino acid sequence is that it predicts a C-peptide of 31 amino acids, 8 residues longer than that reported by Peterson et al. (Peterson, J. D., Nehrlich, S., Oyer, P. E., and Steiner, D. F. (1973) J. Biol. Chem. 247, 4866-4871). The additional octapeptide sequence, Glu-Val-Glu-Asp-Leu-Gln-Val-Arg, is located NH2-terminal to the 23-residue C-peptide sequence described in the earlier report. Its coding sequence is interrupted by the second intervening sequence. The arginine at position 8 suggests that a trypsin-like cleavage may separate the NH2-terminal octapeptide from the remainder of the C-peptide during the post-translational processing of dog proinsulin in the pancreas. The revised C-peptide sequence suggests that the proinsulin C-peptide is more highly conserved in length and overall sequence than was previously supposed.