Characterisation of chicken erythroid nuclear proteins which bind to the nuclease hypersensitive regions upstream of the beta A- and beta H-globin genes.

Chicken erythrocyte sequence-specific nuclear DNA-binding proteins, which bind to the 5'-flanking DNAseI hypersensitive sites of the erythrocyte chromosomal beta A- and beta H-globin genes, have been fractionated by HPLC gel filtration. Three beta A-globin gene DNA binding activities (to sites A, B and B' (10-12)) were separated. The erythroid precursor cell line HD3 has beta A-globin gene sites B and B' binding activities, but binding to site A is detected only after the HD3 cells are induced to differentiate. The fractionated protein binds to a redefined site B', which contains at its center the globin CACCC consensus sequence. The chromosomal beta H-globin gene has two 5'-flanking DNAseI hypersensitive sites which bracket two sequences (H and H') bound by erythrocyte and HD3 nuclear protein in vitro. The beta H- and beta A-globin gene binding sites (H and B) contain variants of the sequences bound by Nuclear Factor 1 and the TGGCA-binding protein, and their protein binding activity(ies) co-purify after HPLC gel filtration.     

DNA sequence alterations affect nucleosome array formation of the chicken ovalbumin gene

The role of the large amount (more than half of the genome) of noncoding DNA in higher organisms is not well understood. DNA evolved to function in the context of chromatin, and the possibility exists that some of the noncoding DNA serves to influence chromatin structure and function. In this age of genomics and bioinformatics, genomic DNA sequences are being searched for informational content beyond the known genetic code. The discovery that period-10 non-T, A/T, G (VWG) triplets are among the most abundant motifs in human genomic DNA suggests that they may serve some function in higher organisms. In this paper, we provide direct evidence that the regular oscillation of period-10 VWG that occurs in the chicken ovalbumin gene sequence with a dinucleosome-like period facilitates nucleosome array formation. Using a linker histone-dependent in vitro chromatin assembly system that spontaneously aligns nucleosomes into a physiological array, we show that nucleosomes tend to avoid DNA regions with low period-10 VWG counts. This avoidance leads to the formation of an array with a nucleosome repeat equal to half the period value of the oscillation in period-10 VWG, as determined by Fourier analysis. Two different half-period deletions in the wild-type DNA sequence altered the nucleosome array, as predicted computationally. In contrast, a full-period deletion had an insignificant effect on the nucleosome array formed, also consistent with the prediction. An inversion mutation, with no DNA sequences deleted, again altered the nucleosome array formed, as predicted computationally. Hence, a VWG dinucleosome signal is plausible.     

An equilibrium between distorted and undistorted DNA in the adult chicken beta A-globin gene

We have used single strand specific nucleases to map DNA distortion in the adult chicken beta A-globin gene. We have detected two structures of that kind and have mapped nuclease-cutting sites at one base resolution. One prominent site is centered at -190 relative to the RNA capping site and is positioned at the center of a stretch of contiguous C residues. The second site is near the first intron/exon junction (+620) and appears as a series of discrete 1-base-long enzyme-cutting sites. Based upon the pattern of nuclease cutting and the kinetics of nuclease cutting we conclude that the "poly(C)" stretch may assume a looped geometry in supertwisted DNA molecules which is similar to that proposed by Felsenfeld (Nickol, J. M., and Felsenfeld, G. (1983) Cell 35, 467-477). We show that S1 nuclease cuts within the intron occur mainly at the end points of polypurine segments and suggest that such end points may assume a distorted transitional geometry. We find that Neurospora crassa endonuclease cuts both the promotor and intron sites in linear DNA molecules but that in linear DNA the cutting process is limited by a first order conformation change of the DNA substrate. Based upon those kinetics we propose that in unstressed DNA, each of the two sites can convert between a distorted and undistorted geometry. In the enzyme assay buffer at 37 degrees C, the time constant for the equilibrium is nearly 10 h for the promotor site and 7 h for the intron.     

DNA sequence organization and transcription of the chicken genome

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard ‘Xenopus’ or ‘Drosophila’ patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)⁺ mRNA. Sequences complementary to poly(A)⁺ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.     

DNA cloning and sequence analysis of chicken AFLP

Amplified fragment length polymorphisms (AFLP) have been shown to be useful for linkage mapping in chickens and other domestic animals. It is often desirable to convert AFLP bands to sequence-tagged site (STS) markers, in particular, so that AFLP-based linkage information can be integrated with recombinant DNA clone-based maps. Sixteen chicken AFLP bands were excised from gels, re-amplified, cloned and analysed. All inserts proved to be EcoRI-TaqI fragments, which suggests that unlabelled TaqI-TaqI AFLP fragments do not amplify well, and therefore do not significantly contaminate AFLP bands. For eight of the AFLP, the cloned fragment was used to probe blots of AFLP reaction fingerprints, confirming that the predominant DNA clone indeed contained the polymorphic fragment. Flanking regions of selected AFLP fragments were isolated using Vectorette cloning. The results obtained suggest that the these chicken AFLP most commonly arise from sequence polymorphism at or near the TaqI site.     

Expression and DNA sequence of the cloned bacteriophage T4 dCMP hydroxymethylase gene.

A plasmid expressing the cloned bacteriophage T4 gene 42 gave the same levels of complementation of gene 42 mutants in a polarity-suppressing rho mutant as in a rho+ host. A reading frame likely corresponding to gene 42 and putative promoter and terminator sequences were identified in the partial sequence of the cloned fragment.     

Nucleotide sequence of a chicken delta-crystallin gene.

We have determined the complete nucleotide sequence of one of the two non-allelic delta-crystallin genes in the chicken, arbitrarily designated delta-gene 1, using a genomic clone (lambda g delta 106) containing the entire gene sequence. By comparison of the genomic sequence and the delta-crystallin cDNA sequence previously determined, we have identified exon sequences in the genomic sequence. Thus, the presence of 17 exons and 16 introns in the gene has been clarified. The delta-crystallin polypeptide deduced from the exon sequences consists of 465 amino acids which is larger, by 19 amino acid residues, than the polypeptide deduced from the cDNA sequence previously reported. Re-examination of the cDNA sequence using the same cDNA clone previously used shows that the present exon sequences are correct and the molecular weight of the deduced delta-crystallin polypeptide is 50,615 daltons instead of the previously reported value of 48,447 daltons. In addition, some structural features of the delta-crystallin gene including putative expression signals are discussed.     

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.     

Complementary DNA sequence of lamprey fibrinogen beta chain

The cDNA sequence of the beta chain of lamprey fibrinogen has been determined. To that end, an oligonucleotide probe was synthesized that corresponded to an amino acid sequence from the carboxy-terminal region of the lamprey fibrinogen beta chain. The insert actually began with residue 3 of the fibrin beta chain; it ran through to a terminator codon following the carboxy-terminal residue at position 443 and then continued for an additional 606 nucleotides of noncoding sequence to its 3' end. The inferred amino acid sequence was verified by comparison with assorted cyanogen bromide fragments isolated from the beta-chain protein, including two carbohydrate-containing peptides that corresponded to segments containing the carbohydrate-attachment consensus sequence. Overall, the lamprey chain is 49% identical with the beta chain from human fibrinogen. This is the same degree of resemblance as was found for the lamprey and human gamma chains. Moreover, the principal regions of conservation are the same in both the beta and gamma chains. Differences and similarities in the physiological behavior of the two fibrinogens are assessed in terms of the observed amino acid replacements.     

Organization, sequence and nuclease hypersensitivity of repetitive elements flanking the chicken apoVLDLII gene: extended sequence similarity to elements flanking the chicken vitellogenin gene.

Analysis of nuclease hypersensitivity of regions flanking the estrogen-dependent, chicken apoVLDLII gene has revealed an hepatic, DNaseI hypersensitive site whose sensitivity is influenced by both the developmental stage and sex of the bird. The site is located 3.0kb upstream from the gene, in a block of middle repetitive elements. Contact hybridization studies indicate that the block consists of contiguous copies of two elements with reiteration frequencies of 500-1000 and 10,000-30,000 copies per haploid genome. Sequencing of 1.8kb spanning the repeats has revealed that the higher frequency element is a member of the CR1 family. The adjacent lower frequency repeat can also be found next to another member of the CR1 family located in the 3' flanking region of the vitellogenin gene. The hypersensitive site has been mapped to one of the two most highly conserved regions of the CR1 element. This region displays homology with a silencer sequence recently identified in a CR1 element flanking the chicken lysozyme gene.     

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.     

Expression of the chicken transferrin gene in transgenic mice

The chicken transferrin gene was microinjected into the male pronucleus of fertilized mouse eggs, and the eggs were then implanted into foster mothers. Approximately 15%-30% of the offspring from the injected eggs carried chicken DNA sequences; restriction mapping indicated that multiple copies of the chicken gene had integrated into the genome in a tandem arrangement in most of the mice. Six of the seven mice studied expressed the chicken gene, and in five mice there was a 5 to 10 fold preferential expression of chicken transferrin mRNA in liver compared to that in other tissues. Chicken transferrin was secreted into the serum of five of the mice, where it reached steady state concentrations up to 67 micrograms/ml. Offspring from transgenic parents also expressed the chicken gene; in some cases the expression in offspring was very similar to the parent, but in one line expression in offspring had increased 2 to 4 fold.     

Structure and partial genomic sequence of the human retinoblastoma susceptibility gene

This report describes the genomic organization of the human retinoblastoma susceptibility locus. This gene spans approximately 200 kb of DNA within human chromosome 13, band q14. The previously determined cDNA sequence comprises 27 exons, ranging in size from 31 bp to 1873 bp, and 26 introns, ranging in size from 80 bp to 70,500 bp. We have mapped the positions of the exons and the positions of the recognition sites for six restriction endonucleases. We also present the sequence of 9.2% of the locus (18,335 bp), including approximately 200 bp of intron sequence immediately flanking each exon. This map of a wild-type allele will form the foundation for future studies of mutant, oncogenic alleles at this locus.     

Expression and purification of chicken beta interferon and its antivirus immunological activity

Interferon beta (IFN-β) belongs to a class of natural proteins that can inhibit virus replication. In this paper, nucleic acid sequence encoding chicken interferon beta (chIFN-β) mature protein was cloned and highly expressed in Escherichia coli (E. coli) by 1mM IPTG induction. The expressed chIFN-β was about 20% of total bacterial protein. The recombinant protein in form of inclusion body was then solubilised, refolded and purified to a purity of greater than 95% by immobilized metal ion affinity chromatography (IMAC). This recombinant chIFN-β could inhibit vesicular stomatitis Indiana virus (VSV) and infectious bursal disease virus (IBDV) replication in vitro. Animal experiments showed that the recombinant chIFN-β could decrease pathological lesions caused by the IBDV in bursa of Fabricius. These results will provide useful information for further investigations on veterinary clinical applications and fundamental research.