Chicken lysozyme gene contains several intervening sequences.

The organization of the chicken lysozyme gene and its neighboring sequences was examined by a comparison of the restriction map of the lysozyme structural gene with the map of the lysozyme gene in genomic DNA. Chicken DNA was cleaved with restriction endonucleases and the DNA fragments were separated by agarose gel electrophoresis. After transfer of the fragments onto nitrocellulose filters, those fragments that contain lysozyme mRNA sequences were detected by hybridization of the filters to labeled probes generated from pls-1, a recombinant plasmid carrying the lysozyme structural gene. This analysis revealed the presence of at least three intervening sequences, two of which interrupt the protein coding region and one of which is located in the 3' untranslated region. When oviduct DNA and sperm DNA were compared, no difference was observed in the size and number of restriction fragments that contain either lysozyme or ovalbumin structural gene sequences.     

The ovalbumin gene: Cloning of the natural gene

The structural ovalbumin DNA sequences are not contiguous and are separated by multiple "intervening regions" in native chicken DNA. EcoRI, a restriction endonuclease that does not cleave the structural ovalbumin DNA sequences, digests the natural ovalbumin gene into three distinct fragments of 2.4, 1.8, and 9.5 kilobase pairs in length by cleaving within these "intervening regions." The 2.4-kilobase pair fragment contains only about 450 nucleotide pairs of coding sequence, with the rest being intervening sequences. This DNA fragment was cloned in bacteria by using the certified EK2 vector lambdagtWES.lambdaB after enrichment from total EcoRI-digested chicken DNA by a combination of RPC-5 column chromatography and preparative agarose gel electrophoresis. Five out of approximately 20,000 recombinant phage plaques were capable of hybridizing with a (32)P-labeled Hha I fragment of a recombinant plasmid pOV230 containing the entire structural ovalbumin gene. DNA amplified in these recombinant phages, lambdagtWES.OV2.4, was shown to contain the same restriction endonuclease cleavage sites as in the 2.4-kilobase pair EcoRI fragment previously determined by restriction mapping of total genomic chicken DNA. The intervening sequences were allowed to hybridize with excess total chicken DNA and oviduct nuclear RNA after nick-translation. They were found to be unique chicken DNA sequences, and appeared to be transcribed in their entireties during gene expression. Like the structural gene sequences, the expression of the intervening sequences is also inducible by steroid hormones.     

The ovalbumin gene: structural sequences in native chicken DNA are not contiguous.

The sequence organization of the structural ovalbumin gene and flanking sequences in native chicken DNA was studied by restriction mapping and filter hybridization using a nick-translated probe generated from pOV230, a recombinant plasmid that contains a full-length ovalbumin DNA synthesized from ovalbumin mRNA. The structural sequences of the ovalbumin gene in native chicken DNA were found to be noncontiguous because at least two restriction endonucleases that do not cut the structural sequence do cleave the natural gene into multiple fragments by cleaving within nonstructural sequences interspersed between the structural sequences. The observation that all ovalbumin DNA-containing sequences were contained within a single DNA fragment generated by BamHI digestion of total chicken DNA has allowed us to construct an inclusive restriction map of the natural ovalbumin gene which contains at least two "insert regions." These regions may be further subdivided into alternating structural and insert sequences. Both insert regions were located within the peptide-coding regions of the gene and the sizes of these insert regions were estimated to be approximately 1.0 and 1.5 kilobase pairs, respectively.     

Genomic structure and possible retroviral origin of the chicken CR1 repetitive DNA sequence family.

We have analyzed the sequence and structure of three CR1 family repetitive elements found in the region adjoining the 3' end of a chicken calmodulin gene. Members of this family are approximately equal to 300 base pairs long and are dispersed throughout the chicken genome. The present data, when taken together with that from four CR1s sequenced previously, reveal that the CR1 family has an overall structure possessing several features associated with the long terminal repeats of avian retroviruses. This finding implies that a retroviral mechanism may be responsible for the dispersion of CR1 sequences throughout the chicken genome. The seven different CR1 repeats that have been analyzed exist at defined locations in the chicken genome relative to nearby structural genes. A directional polarity has been assigned to the CR1 family based upon limited sequence homology to mammalian Alu-type sequences. Interestingly, whether present in 5' - or 3'-flanking DNA, the CR1 sequences have an inverse orientation such that they all "point toward" the nearby structural genes. This is consistent with the previously proposed concept that chicken CR1 sequences may be involved in defining the boundaries of active chromosomal domains of gene expression.     

A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals.

We describe a conserved family of bacterial gene products that includes the VirB1 virulence factor encoded by tumor-inducing plasmids of Agrobacterium spp., proteins involved in conjugative DNA transfer of broad-host-range bacterial plasmids, and gene products that may be involved in invasion by Shigella spp. and Salmonella enterica. Sequence analysis and structural modeling show that the proteins in this group are related to chicken egg white lysozyme and are likely to adopt a lysozyme-like structural fold. Based on their similarity to lysozyme, we predict that these proteins have glycosidase activity. Iterative data base searches with three conserved sequence motifs from this protein family detect a more distant relationship to bacterial and bacteriophage lytic transglycosylases, and goose egg white lysozyme. Two acidic residues in the VirB1 protein of Agrobacterium tumefaciens form a putative catalytic dyad, Each of these residues was changed into the corresponding amide by site-directed mutagenesis. Strains of A. tumefaciens that express mutated VirB1 proteins have a significantly reduced virulence. We hypothesize that many bacterial proteins involved in export of macromolecules belong to a widespread class of hydrolases and cleave beta-1,4-glycosidic bonds as part of their function.     

The ovalbumin gene: cloning and molecular organization of the entire natural gene.

We report the analyses of recently cloned restriction fragments of the natural ovalbumin gene that overlap in part with previously cloned DNA fragments but extend further into the flanking sequences of the gene. These clones now permit us to identify the DNA sequence that codes for the 5' end of ovalbumin mRNA. Based on these and previous results, the molecular organization of the entire ovalbumin gene was established. The entire gene is composed of eight structural DNA sequences separated by seven intervening sequences that are not present in the mature mRNA. In addition, an ovalbumin gene clone has been obtained from a chicken gene library. Analysis of DNA isolated from this particular clone by molecular hybridization and electron microscopic mapping revealed that it contains the entire ovalbumin gene a single segment of DNA and its structure was consistent with that predicted from our physical map constructed from individually cloned fragments of the gene.     

Gene sharing by delta-crystallin and argininosuccinate lyase.

The lens structural protein delta-crystallin and the metabolic enzyme argininosuccinate lyase (ASL; L-argininosuccinate arginine-lyase, EC 4.3.2.1) have striking sequence similarity. We have demonstrated that duck delta-crystallin has enormously high ASL activity, while chicken delta-crystallin has lower but significant activity. The lenses of these birds had much greater ASL activity than other tissues, suggesting that ASL is being expressed at unusually high levels as a structural component. In Southern blots of human genomic DNA, chicken delta 1-crystallin cDNA hybridized only to the human ASL gene; moreover, the two chicken delta-crystallin genes accounted for all the sequences in the chicken genome able to cross-hybridize with a human ASL cDNA, with preferential hybridization to the delta 2 gene. Correlations of enzymatic activity and recent data on mRNA levels in the chicken lens suggest that ASL activity depends on expression of the delta 2-crystallin gene. The data indicate that the same gene, at least in ducks, encodes two different functions, an enzyme (ASL) and a structural protein (delta-crystallin), although in chickens specialization and separation of functions may have occurred.     

Differences Between the Integration of Avian Myeloblastosis Virus DNA in Leukemic Cells and of Endogenous Viral DNA in Normal Chicken Cells

The nature of integrated viral DNA in normal and leukemic chicken cells has been studied by sequential nucleic acid hybridization procedures that localize the viral specific DNA in cellular DNA regions differing in reiteration frequency. First, DNA.DNA reassociation was employed to fractionate cellular DNA sequences according to their reiteration frequencies. Next, the DNA in each fraction was denatured, immobilized on nitrocellulose filters, and then hybridized with viral [(3)H]RNA. In normal cells, endogenous viral DNA appears to be associated with cell sequences reiterated 1200 times, and each integration unit appears to have a maximal size approximately equivalent to the 35S RNA subunit of the virion. In infected cells, additional viral sequences are found which reassociate as if they integrated adjacent to unique cellular DNA, or in tandem with endogenous viral DNA.     

Ovalbumin is synthesized in mouse cells transformed with the natural chicken ovalbumin gene.

The entire chicken ovalbumin gene, accompanied by genomic DNA sequences flanking both termini of the gene and three copies of the herpes simplex virus thymidine kinase gene, has been cloned in plasmid pBR322. This recombinant plasmid was linearized and used to transform thymidine kinase-deficient mouse cells. Thymidine kinase-positive transformants were selected by their ability to grow in the hypoxanthin/aminopterin/thymidine (HAT) medium. The entire ovalbumin gene integrated into high molecular weight DNA within all the transformants and retained its original sequence organization. In all of the transformants examined, a protein identified as chicken ovalbumin by immunoreactivity was detected within the cells. It is estimated that between 1000 and 100,000 molecules of chicken ovalbumin were produced per mouse cell in each of these transformants. Our results demonstrate that the mouse cellular machinery can be utilized to accurately express genetic information encoded in a cloned gene from a different eukaryotic organism into its specific protein product.     

Isolation and characterization of overlapping genomic clones covering the chicken alpha 2 (type I) collagen gene.

A series of overlapping recombinant clones, which cover the alpha 2 (type I) collagen gene, have been isolated by stepwise screening of two libraries of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned cDNA containing alpha 2 collagen DNA sequences as hybridization probe. The other clones were obtained by a sequence of screenings using defined fragments of the successive genomic clones as hybridization probes. Several types of experiments indicated that the DNA of these clones are truly overlapping and span 55 kilobase pairs of contiguous DNA sequences in the chicken genome. Sequence analysis of small DNA segments of some of these clones confirm that they contain coding sequences which specify alpha 2 collagen. Electron microscopic analysis of hybrids between type I alpha 2 collagen mRNA and the overlapping genomic clones indicates that the chicken alpha 2 collagen gene has a length of at least 37 kilobases, about 7.4 times longer than the corresponding translatable cytoplasmic mRNA. The coding information for alpha 2 collagen is distributed in more than 50 coding sequences which are interrupted by intervening sequences of various sizes. The structure of the gene implies that the conversion of precursor RNA to mature mRNA for alpha 2 collagen includes at least 50 splicing events.     

Exon cloning: immunoenzymatic identification of exons of the chicken lysozyme gene.

A 10-kilobase DNA fragment containing exons 1 and 2 of the chicken lysozyme gene has been randomly cleaved with DNase I. After tailing and cloning into the plasmid pUK230, Lac+ colonies were selected. Colonies harboring expressed fragments of the exons could be detected by an immunoenzymatic assay using antibodies against lysozyme. The smallest fragment coded for 10 amino acids and the largest coded for almost all residues of exon 2. These results suggest that any gene of any genome cloned in this way can be detected if antibodies against the gene product are available.     

Structure of the gene for the liver cell adhesion molecule, L-CAM.

The liver cell adhesion molecule, L-CAM, mediates calcium-dependent cell-cell adhesion in early embryos and in nonneural epithelia in adult tissues. Earlier studies of cDNAs for chicken L-CAM established the amino acid sequence of the mature protein. The sequence has now been extended in the 5' direction through the precursor and signal sequences and past a consensus translation initiation site. The combined cDNAs were used to isolate genomic clones covering the entire L-CAM coding sequence. The structural gene for chicken L-CAM contains 16 exons ranging in size from 115 to over 1045 base pairs with an average size of 222 base pairs. Single exons do not correspond to known structural elements such as the signal sequence, precursor segment, internal repeats, or membrane-spanning region of L-CAM. Hybridization of restriction digests of chicken genomic DNA with cDNA and genomic probes indicated that there is a single L-CAM gene in the chicken. In contrast to genes for other cell-cell or cell-substrate adhesion molecules, there is no evidence for alternative splicing of exons in this gene.     

Structural studies on oncornavirus-related sequences in chicken genomic DNA: two-step analyses of EcoRI and Bgl I restriction digests and tentative mapping of a ubiquitous endogenous provirus digests and tentative mapping of a ubiquitous endogenous provirus.

DNA from a variety of uninfected chicken cell types has been analyzed by using restriction endonuclease digestion and RPC-5 ion-exchange chromatography followed by agarose gel electrophoresis. Endogenous retrovirus sequences were detected by using a 32P-labeled avian leukosis viral RNA probe. One simple pattern was identified in an individual containing unexpressed endogenous proviral genes (gs-chf-phenotype for group-specific antigens and chicken helper factor) that was common to all individuals studied. A tentative restriction has been derived for this and one other gs-chf-endogenous provirus. Other gs-chf-individuals and individuals with other phenotypes (e.g., gs+ chf+ and gsl chlfhE) showed more complicated patterns that often included additional bands and thus probably additional proviruses. RNA from an avian sarcoma virus was used to detect cellular sequences (sarc) homologous to the viral transforming gene (src). Results have revealed that a single restriction endonuclease EcoRI fragment of 13 x 10(6) daltons contains the majority of these sequences and confirm that they are not adjacent to the endogenous provirus.     

Quantitative and qualitative differences in DNA complementary to avian myeloblastosis virus between normal and leukemic chicken cells.

Hybridization of avian myeloblastosis virus (AMV) RNA with DNA immobilized on filters or in liquid with a vast DNA excess was used to measure the viral specific DNA sequences in chicken cells. Newly synthesized viral DNA (v-DNA) appears within an hour after infection of chicken embryo fibroblasts (CEF) with avian oncornaviruses. A fraction of newly synthesized v-DNA becomes integrated into the cellular genome and the remainder gradually disappears. A covalent linkage between v-DNA and cellular DNA was demonstrated to exist in CEF and in leukemic myeloblasts by alkaline sucrose velocity sedimentation. Hybridization of AMV RNA in DNA excess has revealed that there are 2 clases of viral specific sequences within normal as well as in leukemic cells. The 2 types of sequences differ in their rate of hybridization. The amount of both types of DNA sequences is about 2 times higher in leukemic cells than in normal cells. Both the fast- and slowly reacting sequences in leukemic cells exhibit a higher Tm (2 degrees C) than the respective DNA sequences in normal cells. Furthermore, when nucleotide sequences in AMV RNA complementary to normal DNA are removed first by exhaustive hybridization with normal DNA, the residual RNA only hybridizes with leukemic DNA but not with normal DNA. These results suggest that leukemic cells contain viral specific DNA sequences which are absent in normal cells. Endogenous v-DNA has been shown to be integrated in cellular DNA region(s) with a reiteration frequency of approximately 1,200 copies per cell and each integration unit appears to have a size approximately equivalent to the 35S RNA subunit of the viral genome. Viral sequences acquired after infection appear to be integrated in the unique region of cell DNA, or in tandem with the endogenous viral sequences.     

Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in DNA of uninfected vertebrates.

We have detected nucleotide sequences related to the transforming gene of avian sarcoma vius (ASV) in the DNA of uninfected vertebrates. Purified radioactive DNA (cDNAsarc) complementary to most of all of the gene (src) required for transformation of fibroblasts by ASV was annealed with DNA from a variety of normal species. Under conditions that facilitate pairing of partially matched nucleotide sequences (1.5 M NaCl, 59 degrees), cDNAsarc formed duplexes with chicken, human, calf, mouse, and salmon DNA but not with DNA from sea urchin, Drosophila, or Escherichia coli. The kinetics of duplex formation indicated that cDNAsarc was reacting with nucleotide sequences present in a single copy or at most a few copies per cell. In contrast to the preceding findings, nucleotide sequences complementary to the remainder of the ASV genome were observed only in chicken DNA. Thermal denaturation studies of the duplexes formed with cDNAsarc indicated a high degree of conservation of the nucleotide sequences related to src in vertebrate DNAs; the reductions in melting temperature suggested about 3--4% mismatching of cDNAsarc with chicken DNA and 8--10% mismatching of cDNAsarc with the other vertebrate DNAs.     

Avian carcinoma virus MH2 contains a transformation-specific sequence, mht, and shares the myc sequence with MC29, CMII, and OK10 viruses.

Avian carcinoma virus MH2 has been grouped together with MC29, CMII, and OK10, because all of these viruses share a transformation-specific sequence termed myc. A 5.2-kilobase (kb) DNA provirus of MH2 has been molecularly cloned. The complete genetic structure of MH2 is 5''-delta gag(1.9-kb)-mht(1.2-kb)-myc(1.3-kb)-delta env(?) and noncoding c-region (0.2-kb)-3''. delta gag, delta env, and c are genetic elements shared with nondefective retroviruses, whereas mht is a unique, possibly MH2 transformation-specific, sequence. Hybridizations with normal chicken DNA and cloned chicken c-myc DNA indicate that the mht sequence probably derives from a normal cellular gene that is distinct from the c-myc gene. The genetic structure of MH2 suggests that the delta gag and mht sequences function as a hybrid gene that encodes the p100 putative transforming protein. The myc sequence of MH2 appears to encode a second transforming function. Therefore, it seems that MH2 contains two genes with possible oncogenic function, whereas MC29, CMII, and OK10 each carries a single hybrid delta gag-myc transforming gene. It is remarkable that, despite these fundamental differences in their primary structures and mechanisms of gene expression, MH2 and MC29 have very similar oncogenic properties.