The ovalbumin gene: alleles created by mutations in the intervening sequences of the natural gene.

Two allelic forms of the natural chicken ovalbumin gene have been independently cloned. These alleles differ from each other by an Eco RI restriction cleavage site in one of the seven intervening sequences within the natural ovalbumin gene. Restriction endonuclease mapping and sequence analyses of these cloned genotypic alleles have shown identical sequence organization and molecular structures of the interspersed structural and intervening sequences except for the particular Eco RI cleavage site. Sequencing data of the cloned DNA suggest that this Eco RI site may be created or eliminated by a single base mutation in the intervening sequence of the ovalbumin gene. The occurrence of apparent homozygous and heterozygous allelic forms of the ovalbumin gene in individual hens and roosters within the same breed has been observed. 10 and 40% of the chickens examined are homozygous for the ovalbumin gene with and without the extra Eco RI site, respectively, while 50% of them are heterozygous. Further analysis of individual chicken DNA cleaved by restriction endonuclease Hae III has revealed that there may be a series of such mutational variations within the ovalbumin gene. We have identified two Hae III cleavage sites that do not occur in all of the chickens, thus giving rise to several additional allelic variations of the ovalbumin gene. At least one of these Hae III sites is situated in the intervening sequence of the ovalbumin gene, and its lcoation has been mapped. Such allelic variations must be taken into consideration when determining eucaryotic gene structure by restriction mapping of the genomic DNA. Furthermore, this type of mutation within the intervening sequences of an eucaryotic gene has no known phenotypic manifestation. It represents an extrastructural silent mutation that must be taken account of in studies to estimate the rates of eucaryotic gene sequence divergence during evolution.     

Regulated expression of the chicken ovalbumin gene in a human estrogen-responsive cell line

To study the regulation of expression of the chicken ovalbumin gene by steroid hormones, the entire ovalbumin gene and its flanking sequences were cloned together with the bacterial gene for xanthine-guanine phosphoribosyltransferase in plasmid pBR322. This recombinant plasmid was linearized and used to transform an estrogen-responsive breast carcinoma cell line (MCF-7) which was shown to possess estrogen receptors and to be estrogen responsive. Transformants were selected by their ability to grow in a medium containing mycophenolic acid and xanthine. The entire ovalbumin gene was integrated into high molecular weight DNA within all transformants analyzed and it retained its original sequence organization. Ovalbumin mRNA and protein were identified from these transformant cells and they were found to be indistinguishable from the authentic counterparts. An 8- to 10-fold increase in the amount of ovalbumin mRNA was observed to be present in cells cultured in 10(-8)M estradiol. We also constructed a hybrid gene containing the 5'-flanking sequence and the first exon of the ovalbumin gene which was linked to the xanthine-guanine phosphoribosyltransferase gene such that expression of this bacterial gene would be promoted and regulated by the chicken sequences. After introduction of this hybrid gene into MCF-7 cells, we observed that the survival of the transformed cells in our selection medium was highly dependent on the presence of estradiol. Our results indicated that the chicken ovalbumin sequence was expressed properly and was regulated to some extent by estradiol in this heterologous system.     

The ovalbumin gene family: Structure of the X gene and evolution of duplicated split genes

The X, Y and ovalbumin genes, which are found within a 40 kb region of the chicken genome, are all expressed in oviduct under steroid hormone control, and share some sequence homologies. We have now cloned the complete X gene and have analyzed its structure. It codes for two RNA species, X and X′; both are coded by eight exons and appear to differ only by the size of their 3′ untranslated region, X′ RNA being 1400 nucleotides longer than X RNA. The striking similarity in the number and length of the exons which constitute the X, Y or ovalbumin genes establishes that they have evolved from a common ancestor gene by duplication events. Comparison of selected regions of the X and ovalbumin genes indicates that the exon sequences coding for protein and the location of the splice junctions have been well-conserved. The introns and the 3′ untranslated exonic sequences have diverged much more rapidly. Four regions of apparently unrelated repetitive sequences are found both outside the X gene and within it (in two introns and in the sequence coding for the 3′ untranslated part of X′RNA). The intragenic repetitive sequences have no counterpart in the ovalbumin and Y genes.     

Metabolic formation of nucleoside-modified analogues of S-adenosylmethionine

A Pseudo-ovalbumin gene, bearing significant nucleotide sequence homology to the ovalbumin gene, has been cloned from genomic chick DNA. Similar to the authentic ovalbumin gene, the pseudo-gene is a unique sequence gene in the chick genome and is expressed at a low level in the immature chick oviduct. In contrast to the ovalbumin gene, expression of the pseudo-gene in the oviduct is not inducible by estrogen. The concentration of pseudo-gene RNA is only ～0.01% of that of authentic ovalbumin mRNA in estrogen-stimulated oviduct cells. Nucleotide sequence analysis of the two sequence related genes may reveal the molecular basis of differential response to steroid hormone induction in the same tissue.     

DNA organisation in the chicken lysozyme gene region

DNA sequences surrounding the lysozyme gene of the chicken have been cloned in several recombinants which define a region of 40 Kb. We have detected no other gene with a sequence related to that of the lysozyme gene, nor any gene expressed in the oviduct in these recombinants. This situation contrasts with that of the ovalbumin gene, in the vicinity of which lie two other genes of related structure expressed in the oviduct under hormonal control. The lysozyme gene region, however contains a complex array of repeated sequences, which have been resolved into at least five classes. An inverted repeat overlaps the lysozyme gene itself.     

Introns of the chicken ovalbumin gene promote nucleosome alignment in vitro.

A defined in vitro chromatin assembly system was used to examine the nucleosome alignment induced by histone H5 throughout a 12 kilobase pair chicken genomic DNA fragment containing the ovalbumin gene. In contrast with total fragmented chicken DNA and several anonymous cloned fragments, much of the gene permitted histone H5 to space nucleosomes at physiological intervals in an extended array. Nucleosomes at the 3'-end of the gene and on approximately 4 kilobase pairs of 5'-flanking ovalbumin sequence did not become aligned to appreciable extents. Analysis of cloned 2-3 kilobase pair subfragments suggested that a strong nucleosome alignment signal, specifying a 196 +/- 5 base pair repeat exists in intron E. A second discrete region of the gene, which mapped approximately to intron A, exhibited nucleosome alignment with a spacing periodicity of about 200 base pairs. The ovalbumin cDNA did not permit nucleosome alignment. These findings suggest that some of the introns contain signals that direct nucleosome alignment over the ovalbumin gene in a way conducive to its regulation.     

DNase I sensitive domain of the gene coding for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase

We have cloned a 36-kilobase segment of chicken DNA containing the gene coding for glyceraldehyde-3-phosphate dehydrogenase [GAPDH (EC 1.2.1.12)], a glycolytic enzyme which is expressed constitutively in all cell types. Using defined segments of this cloned DNA as probes, we have determined the DNase I sensitive domain of the GAPDH natural gene in the hen oviduct. When nuclei isolated from hen oviduct were treated with DNase I under conditions known to preferentially degrade actively transcribed genes (i.e., 15-20% of the DNA rendered perchloric acid soluble), a region of approximately 12 kilobase(s) (kb) containing the GAPDH coding sequences and flanking DNA was found to be highly susceptible to digestion by DNase I. Approximately 4 kb downstream from the end of the coding sequences, there was an abrupt transition from the DNase I sensitive or "open" configuration to the resistant or "closed" configuration. The chromatin then remained in a closed conformation for at least 10 kb further downstream. On the 5' side of the gene, the transition from a sensitive to a resistant configuration was located about 4 kb upstream from the gene. In addition, we have localized two repeated sequences in the area of DNA that was cloned. One of these is of the CR1 family of middle repetitive elements. It is located about 18 kb 3' to the gene and as such lies well outside of the DNase I sensitive region which encompasses GAPDH. The other repetitive element is of an uncharacterized family. It is located upstream from the gene and appears to be within a region of transition from the DNase I sensitive to resistant states.     

Partial purification of the ovalbumin gene

Cellulose-bound DNA complementary to ovalbumin mRNA was used in a continuous hybridization system to isolate single-stranded DNA molecules containing the ovalbumin gene. Fragmented DNA segments containing the ovalbumin gene were enriched 300–350 fold in one cycle of purification. Two cycles of purification resulted in a DNA fraction which was enriched 2300 fold in the ovalbumin sequence. The method is suitable for purification of the ovalbumin sequence from both sheared DNA fragments, as well as larger molecular weight DNA containing more than twice the number of nucleotides necessary to code for ovalbumin mRNA. The chromatographic procedures were specific and reproducible. In addition, the recovery of ovalbumin DNA was essentially quantitative (80–100%), even when large amounts of starting DNA (70–75 mg) were used. This purification scheme should also be useful for the enrichment of other unique sequence genes from eucaryotic DNA.     

DNA methylation: correlation with DNase I sensitivity of chicken ovalbumin and conalbumin chromatin.

To analyse the relationship between DNA undermethylation at some sites in the ovalbumin and conalbumin gene regions (1) and the expression of these genes in chick oviduct, digestions with HhaI, which differentiates between methylated and unmethylated HhaI restriction sites, was performed on DNA isolated from chicken erythrocyte or oviduct chromatin treated with DNase I which degrades preferentially "active" chromatin. This was followed by analysis with ovalbumin- and conalbumin-specific hybridization probes. We conclude that the residual DNA methylation found at some sites of the ovalbumin and conalbumin gene regions is derived from the fraction of cells in which the chromatin of these genes is not in an "active" form. On the other hand, the ovalbumin and conalbumin sites which are partially unmethylated in erythrocyte DNA correspond to chromatin regions which are not DNase I-senitive. We have also detected a site about 1 kb downstream from the 3' end of the conalbumin gene that is hypersensitive to DNase I in all tissues tested.     

鸡卵清蛋白基因启动子调控GFP基因在鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞的表达

特异性扩增家鸡卵清蛋白基因上游调控序列 1340bp～ +16 5 5bp片段和第一内含子 +49bp～ +16 5 5bp片段 ,去除pG FP N2载体自身的CMV启动子 ,分别构建了P_2.9koval GFP和P_1.5koval GFP两种表达载体 ,经测序和酶切鉴定表达载体构建正确。采用脂质体转染法分别将这两种载体、pGFP N2 (阳性对照 )质粒及阴性对照转染鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞。用荧光倒置显微镜观测绿色荧光蛋白的表达。结果表明 :两种表达质粒在鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞中都可以表达荧光蛋白。结果既显示卵清蛋白第一内含子对基因的表达起到一定的调控作用 ,也显示卵清蛋白启动子对输卵管上皮细胞和卵巢细胞不存在特异性 ,并且不存在种属差异性。     

Searching for coding sequences in the mammalian genome: the H-2K region of the mouse MHC is replete with genes expressed in embryos.

We have searched for expressed genes in 170 kb of cosmid cloned DNA from the H-2K region of the mouse MHC. This region is known to contain two genes, H-2K and K2. We identified unique/low copy sequences evenly spaced along the cloned DNA, and used these as probes to search for conserved sequences in Southern blots from a variety of mammalian species. The majority of the unique sequences were found to have homologues and most of these were associated with CpG non-methylated islands. Northern blot analysis and isolation of clones from 5.5 and 10.5-day embryo cDNA libraries showed five additional genes encoded in the H-2K region. Four of these are abundant in embryos; the fifth is exclusively expressed in lymphoid cells. Our data indicate a minimum of seven genes in 170 kb, an unexpectedly high gene density. These results differ from two recent studies where similar lengths of cloned DNA were examined for expressed genes, and only one, or a part of one gene was found. The combined data suggest that the spatial organization of genes in the mammalian genome may not be random.