A new repetitive element of the CR1 family downstream of the chicken vitellogenin gene.

We have analyzed a repetitive DNA sequence found in the 3'-flanking region of the chicken vitellogenin gene. By its sequence, the repetitive DNA has been identified as a hitherto unreported member of the chicken CR1 family of repetitive elements. The CR1 sequence displays the structural characteristics of a long terminal repeat located at the 3' end of an avian retrovirus. The CR1 element lies 2.2 kb downstream of the vitellogenin gene and 'points' away from the gene rather than toward it. In this respect, this element differs from other CR1 repeats. The CR1 element is embedded in a region showing changes in chromatin structure implying a potential role for this sequence in determining the structural state of the local chromatin.     

CTCF-dependent enhancer blockers at the upstream region of the chicken alpha-globin gene domain

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5′ flanking region of the chicken α-globin domain, 11–16 kb upstream of the embryonic α-type π gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two α-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific α-globin gene domain located in a permanently open chromatin area.     

Complete nucleotide sequence of the chicken alpha A-crystallin gene and its 5' flanking region

The chicken alpha A-crystallin gene and 2.6 kb of its 5' flanking sequence have been isolated and characterized by electron microscopy and sequencing. The structural gene is 4.5 kb long and contains two introns, each approx. 1 kb in length. The first intron divides codons 63 and 64, and the second intron divides codons 104 and 105, as in rodents. There is little indication that the insert exon of rodents (an alternatively spliced sequence) is present in complete form in the chicken alpha A-crystallin gene; small stretches of similarity to this sequence were found throughout the gene. The 5' flanking sequence of the chicken alpha A-crystallin gene shows considerable sequence similarity with other mammalian alpha B-crystallin genes. In addition, one consensus sequence (GCAGCATGCCCTCCTAG) present in the 5' flanking region of the chicken alpha A-crystallin gene was found in the 5' flanking region of most reported crystallin genes.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.     

Identification and sequence analysis of the 5' end of the major chicken vitellogenin gene.

We have precisely determined the positions of the first three exons for the major chicken vitellogenin gene (VTG II) by a combination of S1 nuclease protection, primer extension and DNA sequencing experiments. In addition, we have determined the nucleotide sequences of the 5' flanking nuclease hypersensitive sites that we have previously shown are induced during the estrogen mediated activation of the VTG II gene in liver (1). One of these sites is found to be nearly identical to the enhancer core sequence of SV40. A computer assisted analysis of the DNA sequences upstream from the VTG II gene has revealed four short (7 to 9 base pair) sequence elements that are present in similar positions flanking the other major estrogen inducible gene for liver, very low density apolipoprotein II (apoVLDL II). For VTG II, these sequences are located between two of the induced nuclease hypersensitive sites that are liver specific. Sequences homologous to one element, located approximately 100 base pairs upstream from the mRNA cap sites of the VTG II and apoVLDL II genes, are also observed for three estrogen inducible genes that are expressed in the oviduct, although for each of these genes the sequence falls further upstream, between -220 and -200. We suggest that these conserved sequences may be important in mediating the tissue specific responses of these genes to estrogen.     

Stably bent and low-melting DNA sequences at the origin of replication of chicken alpha-globulin gene domains]

The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site of the alpha-globin gene domain has been studied. The fragment was shown to contain the CR1-type repetitive element and two stable bent DNA sequences.     

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.     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Multiple potential intragenic regulatory elements in the CFTR gene

The CFTR gene exhibits a complex pattern of expression that shows temporal and spatial regulation though the control mechanisms have not been fully elucidated. We have mapped DNase I hypersensitive sites (DHS) flanking the CFTR gene to identify potential regulatory elements. We previously characterized DHS at -79.5 and -20.9 kb with respect to the CFTR translational start site, DHS 3' to the gene at 4574 + 5.4-7.4 and 4574 + 15.6 kb, and a regulatory element in the first intron of the gene at 185 + 10 kb. We generated a cosmid contig to provide probes to evaluate the whole of the CFTR gene for DHS and have now mapped novel sites in introns 2, 3, 10, 16, 17a, 18, 20, and 21. These DHS show different patterns of cell-specific expression.