The rabbit beta-globin gene contains a large large insert in the coding sequence

We have used the rabbit β-globin DNA plasmid PβG1 (Maniatis et al., 1976) labeled with ³²P as a filter hybridization probe for DNA fragments containing the β-globin gene in restriction endonuclease digests of rabbit liver DNA. The β-globin DNA fragments we detect appear to contain the gene, present in PβG1 DNA, which codes for adult rabbit β-globin. These fragments have been ordered into a physical map of cleavage sites within and neighboring the structural gene in the rabbit genome (Jeffreys and Flavell, 1977). A detailed analysis of β-globin DNA fragments produced by cleavage with restriction endonucleases which are known to cut the β-globin gene has now shown that the β-globin structural gene is not contiguous in rabbit liver DNA, but is interrupted by a 600 base pair DNA segment inserted somewhere within the coding sequence for amino acid residues 101–120 of the 146 residue β-globin chain. Otherwise, the map of cleavage sites within the gene is co-linear with that deduced from the sequence of rabbit β-globin messenger RNA. Preliminary analysis indicates that this insert is also present in the β-globin gene in rabbit brain, kidney, spleen, bone marrow and sperm, and in erythroid cells isolated from the marrow of an anemic rabbit. The insert appears, therefore, to be a general property of the rabbit β-globin gene, even in tissues in which this gene is active, which suggests that the insert is not involved in inactivating the gene in nonerythroid tissues.     

Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.     

Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.     

Mimotopes and proteome analyses using human genomic and cDNA epitope phage display

In the post-genomic era, validation of candidate gene targets frequently requires proteinbased strategies. Phage display is a powerful tool to define protein-protein interactions by generating peptide binders against target antigens. Epitope phage display libraries have the potential to enrich coding exon sequences from human genomic loci. We evaluated genomic and cDNA phage display strategies to identify genes in the 5q31 Interleukin gene cluster and to enrich cell surface receptor tyrosine kinase genes from a breast cancer cDNA library. A genomic display library containing 2 x 106 clones with exon-sized inserts was selected with antibodies specific for human Interleukin-4 (IL-4) and Interleukin-13. The library was enriched significantly after two selection rounds and DNA sequencing revealed unique clones. One clone matched a cognate IL-4 epitope; however, the majority of clone insert sequences corresponded to E. coli genomic DNA. These bacterial sequences act as 'mimotopes' (mimetic sequences of the true epitope), correspond to open reading frames, generate displayed peptides, and compete for binding during phage selection. The specificity of these mimotopes for IL-4 was confirmed by competition ELISA. Other E. coli mimotopes were generated using additional antibodies. Mimotopes for a receptor tyrosine kinase gene were also selected using a breast cancer SKBR-3 cDNA phage display library, screened against an anti-erbB2 monoclonal antibody. Identification of mimotopes in genomic and cDNA phage libraries is essential for phage display-based protein validation assays and two-hybrid phage approaches that examine protein-protein interactions. The predominance of E. coli mimotopes suggests that the E. coli genome may be useful to generate peptide diversity biased towards protein coding sequences.ABBREVIATIONS USED: IL, interleukin; ELISA, enzyme linked immunoabsorbant assay; PBS, phospho-buffered saline; cfu, colony forming units.     

A molecular description of telometic heterochromatin in secale species

We report the nucleotide sequence of a rabbit β-globin pseudogene, ψβ2. A comparison of the ψβ2 sequence with that of the rabbit adult β-globin gene, β1, reveals the presence of frameshift mutations and premature termination codons in the protein coding sequence which render ψβ2 unable to encode a functional β-globin polypeptide. ψβ2 contains two intervening sequences at the same locations in the globin protein coding sequence as β1 and all other sequenced β-globin genes. An examination of the DNA sequences at the intron/exon junctions suggests that a putative ψβ2 precursor mRNA could not be spliced normally. We compare the flanking and noncoding sequences of ψβ2 and β1 and discuss the evolutionary relationship between these two genes.     

Evolution of the β-globin gene cluster in man and the primates

The arrangement of primate β-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to man. The arrangement of the entire ?γγδβ-globin gene cluster in the gorilla and the yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequence divergence of approximately 1% in 5 million years. A new world monkey (owl monkey) has a single γ-globin gene, suggesting that the ^Gγ-^Aγ-globin gene duplication in man is ancient, and occurred about 20 to 40 million years ago. The β-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 20,000 base-pairs) and contains single ?-, γ- and β-globin genes. The γ- and β-globin genes in this animal are separated by a curious gene containing the 3′ end of a β-globin gene preceded by sequences related to the 5′ end of the ?-globin gene.     

Isolation of β-globin-related genes from a human cosmid library

A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease Mboi, size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in λ phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150000 recombinant-DNA-containing colonies were screened for the presence of the human β-globin related genes. Five recombinants were isolated containing the human β-globin locus and encompassing approx. 70 kb of human DNA.     

Restriction enzyme analysis of the β-globin gene in DNA from β°-thalassaemic subjects from Ferrara

The map of restriction sites including and surrounding the δ- and β-globin genes has been established for three Ferrara β°-thalassaemic subjects. The fragments obtained using nine restriction enzymes do not show any differences from normal DNA. Among others, restriction enzymes giving short fragments at the 5′ and 3′ ends of the β-globin structural gene have been employed. The results obtained for the thalassaemic DNA are identical to those for control DNA, thus excluding the presence of extensive deletions in or adjacent to the coding regions of the β-globin gene in Ferrara β°-thalassaemia.     

Hb switching in chickens

We have taken advantage of the preferential digestion of active genes by DNAase I to investigate the chromosomal structure of embryonic and adult β-globin genes during erythropoiesis in chick embryos, and in particular to examine the question of hemoglobin switching during development. DNA in isolated red cell nuclei was mildly digested with DNAase I to about 10–15 kb, purified and restricted with a variety of restriction enzymes. The DNA was then separated on agarose gels, transferred to nitrocellulose filters and hybridized with an adult-specific β-globin cDNA clone or a genomic clone containing the genes coding for both an embryonic and an adult β-globin chain. Preferential sensitivity of the respective globin genes was monitored by the disappearance of specific restriction bands after DNAase I digestion of nuclei. In embryonic red cells, both adult and embryonic β-globin genes are very sensitive to DNAase I; however, in adult erythroid lines, the embryonic β-globin gene becomes relatively more resistant but the adult gene remains highly sensitive. Controls showed that all globin genes were resistant to DNAase I in brain nuclei and nuclei from lymphoid cells. Thus the switch from embryonic to adult globin expression is associated with an apparent change in the chromosome structure of the embryonic globin gene as reflected in the gene becoming less accessible to DNAase I in adult red cell nuclei. Our results also show that the chromosomal structure of both adult and embryonic genes is altered in embryonic red cell nuclei; thus the nonexpressed globin gene (that is, the adult gene in embryonic red cells) has already been “recognized” to some degree and marked by the erythroid compartment. The sensitivity of the adult globin gene in embryonic cells may represent a “pre-activation” state of the chromosome.     

An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends

For the preparation of gene libraries, DNA from lambda EMBL3 phage was digested with SalI and EcoRI, and the cohesive ends partially filled-in by addition of dTTP, dCTP and Klenow fragment of DNA polymerase I (PolIk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and PolIk. The phage and genomic DNAs were then mixed and ligated. The recombinant DNAs were packaged in vitro. The efficiency of packaging was 10(5)-10(6) of infectious phage lambda particles per microgram of the genomic DNA (as compared to approx. 10(7) per microgram for the wild-type lambda DNA). This procedure is very rapid and requires only microgram quantities of genomic DNA for preparing an entire gene library. The other important advantage is that multiple independent insertions of genomic DNA cannot occur in a single recombinant phage and self-ligation of phage DNA is blocked. It is also applicable for other SalI-containing vectors.     

Chromosomal arrangement of the chicken β-type globin genes

We have isolated the chicken β-type globin genes from a library of chicken DNA-λ Charon 4A recombinant bacteriophage. There are four β-type genes within this segment of the genome; we believe this represents all of the β-type genes of the chicken. The recombinant λCβG1 contains the embryonic ?- and adult β-globin genes. The hatching β^H and embryonic p-globin genes are found in the recombinant λCβG2. Although λCβG1 and λCβG2 do not physically overlap, we present evidence that all four genes are closely linked and transcribed from the same DNA strand. These experiments demonstrate that the chromosomal regions represented by λCβG1 and λCβG2 lie approximately 1.6 kb apart in the chicken genome. A third recombinant λCβG3 extends the genomic locus studied in the vicinity of the β-type globin genes to approximately 39 kb. The physical order of the chicken β-type globin genes within this segment of the chromosome is 5′ … ?-β^H-β-? … 3′. This arrangement is unique among the vertebrate β-type globin gene clusters thus far examined, in that embryonic genes are located at the 5′ and 3′ ends of the cluster while the hatching and adult genes occupy central positions.     

Construction of a gene library using partial filling of DNA sticky ends

To prepare gene libraries, the incomplete filling of protruding ends has been used. DNAs from phages EMBL 3 and EMBL 3a were sequentially digested with SalI and EcoRI, followed by addition of dTTP, dCTP, and DNA polymerase I (Klenow's fragment). Separately, a genomic DNA was partially cleaved with Sau3AI, followed by addition of dATP, dGTP, and Klenow's fragment. The fragmented phage and genomic DNAs were mixed and ligated, and the recombinant DNAs packed in vitro with the phage proteins. The effectiveness of packaging per microgram of genomic DNA was 10(5) to 10(6) (for the wild phage DNA, 10(7)). The proposed procedure is very rapid and needs only microgram quantities of genomic DNA for preparing a representative gene library. It is also useful for other vectors, containing SalI sites.     

GrB deletion of the chorion locus of the silkmoth Bombyx mori. Localization of the left breakpoint and isolation of the deletion junction

In the silkmoth Bombyx mori, choriogenesis occurs through the developmentally controlled deposition of several related classes of chorion proteins onto the oocyte by surrounding follicular cells. In the GrB mutant strain, a distinctive family of proteins (Hc) normally expressed late in choriogenesis, as well as several proteins of middle development specificity, are missing due to the deletion of the corresponding genes from the chorion locus. In addition, a smaller set of proteins normally confined to mid-choriogenesis is found to be prolonged in expression in homozygote mutant but not heterozygote individuals. To elucidate the molecular organization of the chorion locus in the GrB genotype, we scanned a part of the wild-type locus represented by a chromosomal walk of 270,000 bases through library screening and genomic DNA hybridizations using a series of unique probes. A chromosomal clone, GrB4, whose sequences showed the expected characteristics of the deletion junction, was isolated from a partial EcoRI library of mutant genomic DNA. Through comparative hybridizations, mapping and sequencing, the precise location of one of the deletion breakpoints was identified on one of the clones mapping in the characterized part of the wild-type locus. Attempts to locate the other breakpoint in wild-type DNA and to extend the structural characterization past the deletion junction through chromosomal walking were unsuccessful, due to the apparent absence of these sequences from libraries of wild-type and mutant genomic DNA, respectively. Hybridizations of the deletion region on clone GrB4 to cDNA derived from follicular RNA indicate that no gene sequences are directly interrupted by the deletion, and reveal the presence of a gene sequence of unknown function 1000 to 5000 bases to the right of deletion junction.     

Physical mapping of yeast artificial chromosomes containing sequences from the human β-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human β-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult β-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire β-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the β-globin gene cluster.