Rabbit Genomic DNA Research Articles

Molecular cloning and expression of the regulatory (RG1) subunit of the glycogen-associated protein phosphatase

DNA clones encoding the glycogen-binding (RG1) subunit of glycogen-associated protein phosphatase were isolated from rabbit skeletal muscle lambda gt11 cDNA libraries. Overlapping clones provided an open reading frame of 3327 nucleotides that predicts a polypeptide of 1109 amino acids with a molecular weight of 124,257. Northern hybridization of rabbit RNA identified a major mRNA transcript of 7.5 kilobases present in skeletal, diaphragm, and cardiac muscle, but not in brain, kidney, liver, and lung. Southern analysis of rabbit genomic DNA digested with various restriction endonucleases gave rise to a single hybridizing fragment, suggesting that a single gene is present. Expression of the complete RG1 subunit coding sequence in Escherichia coli generated a protein of apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 160,000, similar to the size of the polypeptide detected by Western immunoblot in rabbit skeletal muscle extracts. The RG1 subunit shares significant homology with the Saccharomyces cerevisiae GAC1 gene product which is involved in activation of glycogen synthase and glycogen accumulation. The homology with GAC1 substantiates the role of this enzyme in control of glycogen metabolism. Hydropathy analysis of the RG1 subunit amino acid sequence revealed the presence of a hydrophobic region in the COOH terminus, suggesting a potential association with membrane. This result suggests that the same phosphatase regulatory component may be involved in targeting the enzyme both to membranes and to glycogen.

Human and mouse S-protein mRNA detected in Northern blot experiments and evidence for the gene encoding S-protein in mammals by Southern blot analysis

Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis.

CDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals.

Sperm binding to the egg zona pellucida in mammals is a cell-cell adhesion process that is generally species specific. The guinea pig sperm protein PH-20 has a required function in sperm adhesion to the zona pellucida of guinea pig eggs. PH-20 is located on both the sperm plasma membrane and acrosomal membrane. We report here the isolation and sequence of a full-length cDNA for PH-20 (available from EMBL/GenBank/DDBJ under accession number X56332). The derived amino acid sequence shows a mature protein of 468 amino acids containing six N-linked glycosylation sites and twelve cysteines, eight of which are tightly clustered near the COOH terminus. The sequence indicates PH-20 is a novel protein with no relationship to the mouse sperm adhesion protein galactosyl transferase and no significant homology with other known proteins. The two PH-20 populations, plasma membrane and acrosomal membrane, could arise because one form of PH-20 is encoded and differentially targeted at different spermatogenic stages. Alternatively, two different forms of PH-20 could be encoded. Our evidence thus far reveals only one sequence coding for PH-20: Southern blots of guinea pig genomic DNA indicated there is a single PH-20 gene, Northern blots showed a single size PH-20 message (approximately 2.2 kb), and no sequence variants were found among the sequenced cDNA clones. Cross-species Southern blots reveal the presence of a homologue of the PH-20 gene in mouse, rat, hamster, rabbit, bovine, monkey, and human genomic DNA, showing the PH-20 gene is conserved among mammals. Since genes for zona glycoproteins are also conserved among mammals, the general features of sperm and zona proteins involved in mammalian sperm-egg adhesion may have been evolutionarily maintained. Species specificity may result from limited changes in these molecules, either in their binding domains or in other regions that affect the ability of the binding domains to interact.

Nucleotide sequence of a rabbit genomic DNA encoding mature endothelin-3

Nucleotide sequence of a rabbit genomic DNA encoding mature endothelin-3

Structure of 5′ Regions and Expression of Phenobarbital-Inducible Rabbit Cytochrome P450IIC Genes

To analyze phenobarbital-inducible genes in the P450IIC subfamily, fragments of rabbit genomic DNA containing portions of the P450IIC1 and P450IIC4 genes were isolated and the DNA sequences of the 5'-flanking regions and exons were determined and compared to that of the P450IIC2 gene. The sequences have consensus TATA motifs about 20 bp from the mRNA initiation sites as determined by single-strand nuclease mapping and primer extension. Sequences similar to those for proposed liver-specific regulatory factors, HNF-1 and CCAAT, are found in the P450 5'-flanking regions. A glucocorticoid response element is present in the P450IIC2 gene but is not conserved in P450IIC1 or P450IIC4. Other sequences similar to binding sites for AP-1, the octamer binding proteins, and SPH-1 are present. Except for the TATA motif, none of the potential regulatory sequences was conserved at the same location in the different genes. The P450IIC1 and P450IIC2 genes exhibited high similarity detectable by dot matrix analysis throughout the 700 bp of sequenced 5'-flanking region while the P450IIC4 gene exhibited high similarity to both P450IIC1 and P450IIC2 only for about -150 nucleotides from the RNA initiation site. Southern analysis using probes from the 5' region of the P450IIC4 gene suggests that there are at least three closely related members in the P450IIC4 subgroup in agreement with previous studies. After phenobarbital treatment, levels of P450IIC4 mRNA increased about fourfold, as measured by dot blot hybridization with a P450IIC4-specific oligonucleotide probe or by single-strand nuclease mapping of the 5' end of the mRNA, indicating that the P450IIC4 gene characterized in this study is responsive to phenobarbital. Phenobarbital treatment of rabbits increased in vitro transcription of RNA for both the P450IIC1/2 and P450IIC4 gene subgroups but only slightly and transiently for the constitutive P450IIC3 gene.

Inactivation of rabbit immunoglobulin λ chain variable region genes by the insertion of short interspersed elements of the C family

Two rabbit germ-line genes encoding immunoglobulin lambda light chain V regions were cloned from a rabbit genomic liver DNA library and characterized. One, V lambda 1, is separated by at least 8 kb from any other V lambda gene. The second, V lamdba 4, forms part of a three-gene cluster with two functional V lambda genes recently reported. Both V lambda 1 and V lambda 4 have structural features rendering them pseudogenes. The coding regions have frame-shift mutations which would yield defective protein products; both genes are also interrupted by the insertions of short, interspersed repetitive elements of the C family. In the V lambda 1 gene, the 369-bp insert is located upstream of the gene between the putative TATA box and the leader exon, whereas in gene V lambda 4, the 360-bp insert interrupts the FR2 at codon 48c. In addition, the sequence of the complement-determining region 3 of gene V lambda 1 is very similar to the mouse DSP2.6 sequence.

Cloning and sequencing of two functional rabbit germ-line immunoglobulin V λ genes

A recombinant-phage library of rabbit genomic DNA was screened for immunoglobulin VA genes. Two functional genes, Vλ2 and Vλ3, which are separated by 1.6kb were isolated and sequenced. Both are accompanied by signals required for transcription-translation and the recombination with Jλ genes. The two genes, which are 95 % similar in their framework regions, may be the parents of several cDNAs encoding VA regions. Rabbit Vλ-region diversity is likely to be generated by somatic mutation and V-J junction flexibility. Two cDNA clones encoding λ-light chains may have arisen by gene conversion, exchanging the complementarity-determining regions, CDR3s, of the Vλ2 and Vλ3 genes.

The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation.

The major apoprotein of rabbit pulmonary surfactant is a developmentally and hormonally regulated sialoglycoprotein, Mr congruent to 29,000-36,000 (SP 29-36). In the present study, specific antibodies were used to isolate cloned cDNA inserts for SP 29-36 from a fetal rabbit lung cDNA library in bacteriophage lambda gt11. Two species of cDNA of 1.9 and 3.0 kilobases (kb) in size were isolated that are complementary to two species of mRNA of 2.0 and 3.0 kb which differ primarily in the lengths of their 3'-untranslated regions. The 2.0-kb species of mRNA is approximately 5 times more abundant than the 3.0-kb mRNA. The results of Southern hybridization analysis of rabbit genomic DNA cut with a number of restriction enzymes are indicative that the two mRNA species are encoded by a single gene. The two mRNA species appear to be expressed only in rabbit lung tissue and are coordinately regulated during development in vivo and in vitro; hybridizable SP 29-36 mRNA is first detectable in rabbit lung tissue on day 26 of gestation, increases to a maximum on day 31, and declines somewhat after birth. The 3.0-kb cDNA is comprised of 57 nucleotides of 5'-untranslated region, an open reading frame of 741 nucleotides, and a 3'-untranslated region of 2,165 nucleotides that contains three poly(A)-addition signals. The most 5' of the poly(A) addition signals is utilized in synthesis of the 2.0-kb SP 29-36 mRNA, while the most 3' is utilized in synthesis of the 3.0-kb mRNA. The open reading frame of the 3.0-kb cDNA encodes a protein of 247 amino acids which is highly homologous to the major apoproteins of dog and human pulmonary surfactant. The SP 29-36 cDNAs were utilized to evaluate the effects of dibutyryl cyclic AMP (Bt2cAMP) on the levels of this mRNA in fetal rabbit lung tissue in organ culture. Bt2cAMP caused an induction of SP 29-36 mRNA that was detectable as early as 2 h after its addition to the medium. This inductive effect of Bt2cAMP was blocked when cycloheximide was also present in the medium for greater than or equal to 4 h. Cycloheximide treatment also reduced the levels of SP 29-36 mRNA in control explants; this inhibitory effect on control and Bt2cAMP-treated explants was reversed within 12 h of the removal of cycloheximide from the medium. These findings suggest that a labile protein factor mediates the transcription of the SP 29-36 gene and its induction by Bt2cAMP.

Recent data on the structure of rabbit milk protein genes and on the mechanism of the hormonal control of their expression.

Mammary explants or isolated mammary cells from rabbit have been cultured in the presence of insulin, prolactin and cortisol alone or in combination. The cellular content in alpha s1-casein, beta-casein and whey acidic protein (WAP) mRNA have been evaluated using the corresponding cDNA as probes. In all cases alpha s1-casein mRNA was the most abundant and WAP mRNA the least abundant mRNA. The three genes showed essentially similar dependency towards hormones. Prolactin stimulated mRNA accumulation and insulin and cortisol amplified this stimulation. The induction by prolactin was rapid whereas stimulation by insulin was slower. Fragments of rabbit genomic DNA inserted in lambda phage and containing alpha s1-casein, and WAP genes have been cloned. The primary sequence around the CAP site of the three genes has been established. A comparison of the sequences located upstream from the CAP site shows several striking homologies with the corresponding genes from cow, rat and guinea-pig. This suggests that these sequences participate in the transcriptional control of the genes by hormones. The mechanism involved in the transduction of the prolactin message to milk protein genes in unknown. Using mammary explants in culture, several classical mechanisms of transduction have been examined. Phorbol ester, phorbol -12, 13-dibutyrate (PdiBu) inhibited prolactin action. However, another tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), did not alter prolactin action. Kinase C inhibitor H7 did not prevent prolactin action and did not overcome the inhibition by PdiBu. Kinase C is therefore not essential for the transduction of the prolactin message to milk protein gene. Neomycin, which inhibits phosphatidylinositol hydrolysis by phosphorylase C, prevented prolactin action, whereas other inhibitors of phosphatidylinositol metabolism remained uneffective. Degradation of phosphatidylinositol is therefore likely not an essential step of prolactin action on milk protein genes. Inhibitors of tyrosine kinase and phosphatase exhibited a poor capacity to modify the prolactin response. Hence, transduction mechanisms using tyrosine kinase activity likely cannot account for prolactin action.

Characterization of cDNA coding for the complete light meromyosin portion of a rabbit fast skeletal muscle myosin heavy chain.

Myosin-heavy-chain-specific cDNA clones have been isolated from a cDNA library prepared from hind leg muscle of a 14-day-old rabbit. According to restriction enzyme analysis these can be grouped into at least two, probably three different classes. RNA dot-blot hybridization shows that all of these clones correspond to mRNAs expressed in fast skeletal muscle. The clones of the most abundant form, class I, can be aligned to cover the complete light meromyosin portion of myosin heavy chain. The sequence of the coding and the 3'-untranslated region, together comprising 2143 base pairs, has been determined. The class I clone detects a multigene family of 8-12 members on a Southern blot of rabbit genomic DNA.

Block duplications of a zeta-zeta-alpha-theta gene set in the rabbit alpha-like globin gene cluster.

In order to understand the coordinate regulation between the alpha-like and beta-like globins during the developmental switches in hemoglobin synthesis, we have studied the rabbit alpha-like globin gene family. A cluster of six linked genes arranged 5'-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3' has been isolated as a set of overlapping clones from a library of rabbit genomic DNA. Blot-hybridization analysis of genomic DNA not only confirms this linkage arrangement but also reveals the presence of additional zeta and theta genes. We propose that this gene cluster was generated by a block duplication of a set of alpha-like genes; the proposed duplication unit is zeta-zeta-alpha-theta. Further duplications of a zeta-zeta-theta set are also proposed to have occurred. As expected for a duplicated locus, the rabbit alpha-like gene cluster contains long blocks of internal homology. The Z homology block is about 7.2 kilobase pairs long and contains the zeta genes; the T homology block is about 4.7 kilobase pairs long and contains a theta gene. Surprisingly, both Z and T homology blocks are flanked by a common junction sequence (J) which contains a region very similar to the 3'-untranslated sequence of an alpha-globin gene. Analysis of the J sequences suggests a recombination mechanism by which the alpha gene could have been deleted from the second set of genes in the cluster (zeta 2-zeta 3-theta 2). The relationships among the genes in characterized alpha-like gene clusters in mammals are summarized. The rabbit gene cluster differs from those of other mammals principally in the loss of a gene orthologous to the human psi alpha 1 and in the block duplication of the zeta-zeta-alpha-theta gene set.

Cloning of a rabbit erythroid-cell-specific lipoxygenase mRNA

We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte (immature red blood cell, RBC) lipoxygenase (LOX) mRNA. One cDNA predicts an amino acid (aa) sequence matching exactly the unique N-terminal 30-aa sequence of the purified enzyme. Further, the reticulocyte mRNA, hybrid-selected by this recombinant, can be translated in vitro to give a polypeptide that comigrates with the purified reticulocyte LOX and is recognised by affinity-purified anti-RBC LOX polyclonal antibodies. Southern blotting experiments hybridising the RBC LOX cDNAs available to total rabbit genomic DNA digested with various restriction enzymes gives a fairly simple hybridisation pattern under moderate stringency conditions: moreover, the same pattern is obtained with a cloned fragment of genomic DNA containing the RBC LOX gene. This indicates that the RBC LOX gene is unique in the genome and seems not to be very closely related to the genes encoding the other tissue LOXs. We also show by Northern transfer/hybridisation experiments that the RBC LOX mRNA is expressed only in the red cell lineage but not in white blood cells (bone marrow or spleen) or in other non-erythroid cells tested (e.g., brain and lung).

Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials.

Two Ca 2+ ATPase genes: Homologies and mechanistic implications of deduced amino acid sequences

Rabbit genomic DNA contains two genes that encode Ca 2+ ATPases of fast twitch and of slow twitch (and cardiac) sarcoplasmic reticulum, respectively. The deduced amino acid sequences of the products of the two genes are highly conserved in putative Ca 2+ binding regions, in sectors leading from cytoplasmic domains into transmembrane domains, and in transmembrane helices. A transport mechanism is proposed in which Ca 2+ binds to negatively charged groups on amphipathic stalk sectors, becoming occluded during enzyme phosphorylation by bound ATP. Rotation of the stalk sectors is induced as the energy in the phosphorylated enzyme (E 1P) is utilized in conformational changes leading to the low energy form, E 2P. Rotation leads to disruption of high affinity Ca 2+ binding sites and release of Ca 2+ into a charge-lined membrane channel. Ca 2+ then traverses the membrane by exchange diffusion.

Isolation and nucleotide sequence of the rabbit globin gene cluster psi zeta-alpha 1-psi alpha. Absence of a pair of alpha-globin genes evolving in concert.

A cloned 13.3-kilobase (kb) region of rabbit genomic DNA contains a cluster of alpha-like globin genes arranged 5'-psi zeta-(3.6 kb)-alpha 1-(2.2 kb)-psi alpha-3'. Genomic blot hybridization data show that this is the major alpha-like globin gene cluster in rabbits, although a second alpha-globin gene (alpha 2) is also detected. Repetitive sequences from the C family of short repeats flank the psi zeta gene, and a new repetitive element, the F repeat is located 3' to psi alpha. The sequence was determined for a 4024-base pair (bp) segment that extends from 149 bp 5' to the cap site of alpha 1 to 207 bp 3' to psi alpha. Gene alpha 1 is functional and encodes one of the major allelic variants of rabbit alpha-globin. This gene has very short introns (77 bp in intron 1 and 83 bp in intron 2) and an unusual ATA box in the 5' flanking region (CTTAAA), which does function to promote transcription by RNA polymerase II in a cell-free system. Short (4 or 9 bp) G + C-rich repeats are interspersed throughout the flanking regions and the introns. Gene psi alpha cannot encode a globin polypeptide, and it is probably inactive. This is shown by the absence of a normal globin gene promoter, the replacement of the 5' untranslated sequence by tandem repeats of the sequence GCCCGCCGC, frameshift deletions in exon 2, and the modification of the polyadenylation signal to AGTAAA. The intergenic region between alpha 1 and psi alpha is very G + C rich (65.7% G + C) and contains many short, tandem repeats. Gene psi zeta hybridizes specifically to a human zeta-globin gene probe, but a partial sequence reveals frameshift mutations that probably make psi zeta a pseudogene. Mammalian alpha-globin gene clusters vary in the presence or absence of pseudogenes, and if present, the position of the pseudogene differs in various gene clusters. Among the mammalian alpha-like gene clusters so far analyzed, the rabbit gene cluster is unique in the absence of duplicated alpha-globin genes that are undergoing concerted evolution.

Mosaic structure and mRNA precursors of uteroglobin, a hormone-regulated mammalian gene.

The synthesis of uteroglobin in the rabbit uterus is induced by progesterone and is repressed by estrogen which has an over-riding effect over the inducer. The dual hormonal control system offers an excellent model for studying hormonal regulation of mammalian gene expression. Using a full-length uteroglobin cDNA clone as a specific hybridization probe, recombinant lambda phages containing the entire chromosomal uteroglobin gene have been isolated from a rabbit genomic DNA library. Electronmicroscopic analysis of hybrid molecules formed between the chromosomal uteroglobin gene and uteroglobin mRNA indicated the presence of 2 intervening sequences within this gene. The mosaic structure of the uteroglobin gene has been substantiated by detailed restriction mapping and Southern hybridization. The gene is 3.0 kilobases in length to code for a mature mRNA of 465 nucleotides. Northern hybridization of poly(A)-containing RNA from 4-day-pregnant rabbit uterus with the full-length cDNA clone revealed the presence of uteroglobin mRNA precursors. The size of the largest precursor RNA species detected by the cDNA clone is the same as the entire chromosomal uteroglobin gene. The fidelity of the precursor RNAs was established by their ability to hybridize with specific intervening sequence probes. Thus the entire uteroglobin gene is expressed into primary RNA transcripts, which are subsequently processed into mature mRNA molecules by splicing.

Localization of repetitive and unique DNA sequences neighbouring the rabbit β-globin gene

DNA fragments containing the rabbit β-globin gene can be detected in restriction endonuclease digests of rabbit DNA, using filter hybridizations with a labelled β-globin cDNA plasmid as a probo for the structural gene. Analysis of the reannealing kinetics of these β-globin DNA fragments has been performed by denaturing rabbit DNA and renaturing to various C 0 t ‡ ‡ Abbreviations used: C 0t , initial DNA concentration (mol.1 −)× time (s); kb, bases × 10 −3; cDNA, complementary DNA; bp, base-pairs values, then determining by hydroxyapatite chromatography and subsequent filter hybridization whether a given β-globin DNA fragment has annealed with other DNA via a repetitive DNA tract contained in the fragment, or instead has remained completely singlestranded. This approach has revealed the presence of an intermediate repetitive DNA sequence about 0.6 to 2.4 kb from the 3′ end of the rabbit β-globin structural gene. The presence of this repetitive sequence was confirmed by filter hybridization analysis of rabbit DNA with various DNA fragments isolated from a cloned segment of rabbit genomic DNA containing the β-globin gene. A purified rabbit DNA fragment containing sequences 0.9 to 2.1 kb from the 3′ end of the structural gene was found to hybridize to many DNA fragments in restriction endonuclease digests of rabbit DNA. In situ hybridizations to rabbit metaphase chromosomes showed that copies of this repetitive sequence were present on several chromosomes, and were preferentially localized in centromeric and telomeric regions. In contrast, the remaining section of the rabbit β-globin gene region examined, extending 1.4 kb from the 5′ end of the β-globin gene to 0.9 kb from the 3′ end of the gene and including the intervening sequences within the structural gene, was shown to consist of single copy DNA.

The organization of repetitive sequences in a cluster of rabbit β-like globin genes

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit β-like globin genes (β1–β4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140–400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.

The isolation of structural genes from libraries of eucaryotic DNA

We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of β-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked β-globin genes.