Structure of the rabbit tc-casein encoding gene: expression of the cloned gene in the mammary gland of transgenic mice

The rabbit κ-casein (κ-Cas) encoding gene has been isolated as a series of overlapping DNA fragments cloned from a rabbit genomic library constructed in bacteriophage λEMBL3. The clones harboured the 7.5-kb gene flanked by about 2.1 kb upstream and 9 kb downstream sequences. The cloned gene is the most frequently occurring of two κ-Cas alleles identified in New Zealand rabbits. Comparison of the corresponding domains in rabbit and bovine κ-Cas shows that both genes comprise 5 exons and that the exon/intron boundary positions are conserved whereas the introns have diverged considerably. The first three introns are shorter in the rabbit, the second intron showing the greatest difference between the two species: 1.35 kb instead of 5.8 kb in the bovine gene. Repetitive sequence motives reminiscent of the rabbit C type repeat and the complementary inverted C type repeat were identified in the fourth and first introns, respectively. Transgenic mice were produced by microinjecting into mouse oocytes an isolated genomic DNA fragment which contained the entire κ-Cas coding region, together with 2.1-kb 5′ and 4.0-kb 3′ flanking region. Expression of transgene rabbit κ-Cas mRNA could be detected in the mammary gland of lactating transgenic mice and the production of rabbit κ-Cas was detected in milk using species-specific antibodies. The cloned gene is thus functional.     

Restriction fragment length polymorphism in the 3′ flanking region of the rabbit β1-globin gene

By Southern blot analysis, a restriction fragment length polymorphism in the 3' flanking region of the rabbit beta 1-globin gene was detected. Two alleles, characterized by 9.7- and 12.4-kb BamHI fragments and by 15.3- and 18.0-kb HindIII fragments, have been detected in a small population of White New Zealand rabbits. The long allele is the most frequent (about 70%). The simultaneous changes in the restriction patterns of the two endonucleases and the constant distance between BamHI and HindIII sites in short and long fragments suggest the possibility that the two alleles arise from a rearrangement phenomenon involving a DNA segment 2.7 kb long. In addition, the presence of the two alleles in individuals genetically unrelated to the White New Zealand breed suggests that this polymorphism is widespread.     

Integration and excision of pMC7105 in Pseudomonas syringae pv. phaseolicola: involvement of repetitive sequences.

The site for integration of pMC7105 into the chromosome of Pseudomonas syringae pv. phaseolicola has been mapped to a 2.6-kilobase-pair (kb) Bg/II-EcoRI fragment on this 150-kb indigenous plasmid. Selected excision plasmids resulting from imprecise excision of pMC7105 were used to identify one of the plasmid-chromosome juncture fragments and to characterize the mechanism of recombination from the chromosome. A 14.2-kb BamHI plasmid-chromosome juncture fragment has been identified in pEX8060 (234 kb), an excision plasmid which carries approximately 90 kb of chromosomal sequences to the left of the site of integration. This fragment contains a portion of the 2.6-kb Bg/II-EcoRI fragment as well as chromosomal sequences. Blot hybridization with a probe made from selected fragments of pMC7105 revealed three distinct repetitive sequences, RS-I, RS-II, and RS-III, on this plasmid. The 2.6-kb fragment, to which the site of integration maps, also contains RS-II. Five copies of RS-II are present in pMC7105, and more than 20 copies are present in the chromosome. Eight small excision plasmids were shown to result from recombination among fragments of pMC7105 that contain common repetitive sequences. The results indicate that integration and excision of pMC7105 occur through general recombination at homologous repetitive sequences.     

Molecular basis of five apolipoprotein B gene polymorphisms in noncoding regions

Restriction fragment length polymorphisms (RFLPs) are useful in linkage and clinical association studies of human diseases. In this report, we characterize the molecular basis and frequencies of two new RFLPs, AvaII and BalI, two previously reported RFLPs, HincII and PvuII, and one new sequence polymorphism in the human apolipoprotein B gene. For the AvaII RFLP, the two alleles yield either a 1 kb fragment or 0.7 and 0.3 kb fragments, and have frequencies of 20% and 80%, respectively. The polymorphic site is about 4 kb upstream of exon 1. For the BalI RFLP, the two alleles yield either a 4.9 or 6.2 kb fragment, and have about equal frequencies. The polymorphic site is within an Alu sequence in intron 20, 146 bp 5' to exon 21. The BalI recognition sequence TGGCCA is replaced by TAGCCA. For the HincII RFLP, the two alleles yield either a 1.7 or 1.3 kb fragment and have frequencies of 80% and 20%, respectively. The polymorphic site is in intron 4, 171 bp 3' to exon 4. The HincII recognition sequence GTTAAC, present in the minor allele, is replaced by GTTACC. HincII fragments of 7.4 and 7.0 kb, previously reported for this polymorphism, are the result of partial digestion at the invariant HincII site in intron 3, 334 bp 3' to exon 3. For the PvuII RFLP, the two alleles yield either a 7.5 or 5.5 kb fragment and have frequencies of 96% and 4%, respectively. The polymorphic site is within an Alu sequence in intron 4, 523 bp 5' to exon 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clone banks of the mung bean, pea and spinach chloroplast genomes

All but one of the PstI restriction fragments from mung bean, pea, and spinach chloroplast DNAs have been stably cloned into pBR322. Large fragments (15-54 kb) were cloned at low efficiencies which decreased with increasing fragment length. However, plasmids containing fragments above 25-30 kb were too unstable to be useful. In particular, pBR322 derivatives containing the largest mung bean and spinach fragments (34 kb and 54 kb, respectively) are extremely unstable and rapidly delete parts of the plasmid sequence. The PstI fragments of mung bean chloroplast DNA which cover the 34-kb PstI fragment have been cloned into pACYC177. After a search of several thousand recombinants we were unable to recover a clone containing a 12.2-kb pea chloroplast PstI fragment and suggest that some property of its sequence may be inimical to the cloning process. The identity of the cloned fragments to native chloroplast DNA restriction fragments is demonstrated by restriction analysis and the ability to construct detailed restriction maps of the mung bean and pea chloroplast genomes.     

Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp

Restriction maps of two plasmids encoding parathion hydrolase have been determined. pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp. (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG). Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping. In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA. The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids is evident outside of this region.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Hypobetalipoproteinemia due to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu recombination

We report the molecular defect in an individual with homozygous hypobetalipoproteinemia. A unique TaqI restriction fragment length polymorphism was found in the midportion of the apolipoprotein B (apoB) gene using the genomic probe, pB51. The probe, which identifies TaqI fragments of 8.4 and 2.8 kilobases (kb) in normal individuals, hybridized to a single 11-kb fragment in the proband. The parents of the proband showed all three TaqI fragments, implying that they are heterozygotes for the mutant apoB allele. In this family, the mutant allele cosegregated with low total cholesterol levels and formal linkage analysis gave a decimal logarithm of the ratio score of 3.3 at a recombination frequency of 0. The polymorphic TaqI site was localized to an EcoRI fragment of 4 kb in normal individuals. The corresponding fragment in the proband was 3.4 kb, suggesting a 0.6-kb deletion in the mutant allele. Both the normal 4-kb EcoRI fragment and the mutant 3.4-kb EcoRI fragment were cloned and sequenced. In the normal allele, the 4-kb EcoRI fragment extends from intron 20 to 23. Exon 21 is flanked by Alu sequences that are in the same orientation. The mutant allele had a 694-bp deletion in this region which included a small part of the Alu sequence in intron 20, the entire exon 21, and most of the Alu sequence in intron 21. The polymorphic TaqI site, which lies within the Alu sequence in intron 21, was absent in the proband as a result of the deletion. The deletion of exon 21 results in a frame shift mutation and the introduction of a stop codon. Translation of the encoded mRNA would yield a prematurely terminated protein. This mutant apoB protein would be 1085 amino acids long with the 73 carboxyl-terminal residues out of frame. We postulate that the deletion of exon 21 is the consequence of a crossover event between the Alu sequences in introns 20 and 21 resulting in nonreciprocal exchange between two chromosomes.     

Molecular cloning of a new endoglucanase gene from Clostridium cellulolyticum and its expression in Escherichia coli

Summary Two genes coding for endoglucanase activity in Clostridium cellulolyticum were cloned and expressed in Escherichia coli by using plasmid pUC18. The sizes of two fragments harbouring endoglucanase genes are 4.4 kb and 2.0 kb, respectively. The 2.0-kb fragment was identical with a reported DNA fragment encoding an endoglucanase of C. cellulolyticum. The 4.4-kb fragment was obtained first in this study. Deletion analysis showed that a 1.3-kb portion of the 4.4-kb fragment is necessary for the endoglucanase expression by its own promoter. The 4.4-kb fragment hybridized with several different fragments of the genomic DNA in C. cellulolyticum.Offprint requests to: T. Kodama     

Physical mapping of the Escherichia coli D-serine deaminase region: contiguity of the dsd structural and regulatory genes.

The genes dsdA, dsdO, and dsdC have been located on a 3.0-kilobase pair (kb) fragment of the Escherichia coli chromosome by a combination of techniques. The loci were first cloned onto lambda and various plasmid vectors. dsd hybrid plasmids were then digested with restriction enzymes, and the fragments were recloned to test for the presence of dsdC or dsdA. In one case, a 4.2-kb restriction fragment containing the dsdA operon was used to form a heteroduplex with a well-defined lambda dsd deoxyribonucleic acid. The results show that dsdA, dsdO, and at least 0.6 kb of dsdC are present on this piece of deoxyribonucleic acid. On the basis of the mapping analysis and the molecular weight of D-serine deaminase, 1.9 kb of the 4.2-kb fragment is accounted for by the three dsd loci. We conclude that dsdO and dsdC are contiguous. A detailed dsd restriction map is presented.     

A discordant sibship analysis between beta-NGF and neurofibromatosis.

A new restriction fragment length polymorphism 5' to the beta-nerve growth factor (beta-NGF) gene has been found in proximity to the BglII polymorphism, and both polymorphisms are detectable with an EcoRI 7-kilobase (kb) subclone. Absence of the TaqI recognition site lengthens the 4.3-kb and 1.7-kb hybridizing fragments to 6 kb, and the alleles are in Hardy-Weinberg equilibrium with frequencies of 83% and 17%, respectively. Previous research has suggested that NGF is involved in disseminated neurofibromatosis (NF). We found four informative disseminated NF families with the two beta-NGF polymorphisms and have provided clearcut evidence against beta-NGF gene alteration in these families. If disseminated NF is found to be heterogeneous at a molecular level, more families should be tested to further rule out any role for beta-NGF in this syndrome.     

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb. In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.     

Restriction fragment length polymorphisms in bovine major histocompatibility complex class II ß-chain genes using bovine exon-containing hybridization probes

Restriction fragment length polymorphisms (RFLPs) have been identified in the bovine MHC class II region using five hybridization probes constructed from two bovine genomic clones. Four probes were constructed from a bovine DR beta-like gene, BoLA-DRB2. These included a probe containing the complete beta 1 exon (R2-beta 1), a probe containing the last 129 base pairs of the beta 2 exon (R2-beta 2), a probe containing intron immediately 5' of the beta 2 exon (R2-5' beta 2), and a probe containing the complete transmembrane exon (R2-TM). A fifth probe was constructed from a novel bovine beta-chain gene, BoLA-DIB, and contained the entire TM exon (I1-TM). R2-beta 1 defined very little polymorphism. R2-beta 2 hybridized to several fragments but one or two fragments hybridized much stronger on all Southern blots and it was presumed these corresponded to BoLA-DRB2 fragments. By using R2-5' beta 2 as a probe, these BoLA-DRB2 fragments were confirmed: 6.4 and 2.7-kb Eco RI alleles, 1.7- and 1.5-kb Pvu II alleles, 5.9-, 5.4-, 3.7- and 1.9-kb TaqI alleles, and a non-polymorphic 22.5-kb BamHI fragment. I1-TM identified three alleles with TaqI. To investigate the linkage between the RFLP alleles, 166 offspring of five sires were tested. Complete linkage was found for all RFLPs identified with the BoLA-DRB2 probes. However, the RFLP patterns of 13 calves out of 58 indicated recombination between BoLA-DRB2 and BoLA-DIB.     

An additional restriction fragment length polymorphism at the thyroglobulin gene.

The study was aimed at the screening of human chromosomal DNA for restriction fragment length polymorphism (RFLP) at the human thyroglobulin (hTg) gene locus. The RFLP screening was performed in a typical way. As hybridization probes were used 5 Pst I fragments of hTg cDNA of the total length 5.1 kb pairs cloned in pBR 322. One not described polymorphism was found by using the probe hTg 10, (nucleotides from position 4830 to 5810 in the 3' flanking region of hTg). Restriction enzyme Msp I identified a single two allele polymorphism: A1: 3.5 kb and A2: 2.5 kb. Of 32 unrelated healthy individuals two were homozygous for 3.5 kb, one was homozygous 2.5 kb and 29 were heterozygous for both 3.5 kb. and 2.5 kb. Thus, the frequencies of the 3.5 and 2.5 kb Msp I alleles were 0.52 and 0.48 respectively.     

Haplotypes of the human immunoglobulin lambda IGLV7S1 and IGLV1S1 genes defined by restriction-site polymorphism and insertion/deletion of a 6-kb DNA fragment.

Haplotypes were defined in the human immunoglobulin lambda locus by using three probes--V lambda VII, V lambda A, and V lambda I--hybridized to BamHI, KpnI, EcoRI, and HindIII digests. Four KpnI alleles were described. Two of them, 13 kb and 16 kb, detected with both the V lambda VII and V lambda A probes, were correlated with 4.6-kb and 10.5-kb KpnI fragments, respectively, which hybridize to the V lambda I probe. The two others (17 kb and 24 kb) were detected with the three probes V lambda VII, V lambda A, and V lambda I. Moreover, we show that two of those haplotypes may reflect an insertion of 6 kb between V lambda A and V lambda 1S1. Familial studies were performed to demonstrate the Mendelian inheritance. Our results demonstrate the absence of association between the C lambda alleles and V lambda haplotypes.     

Genome size of Eperythrozoon suis and hybridization with 16S rRNA gene

The genome size of Eperythrozoon suis, an unculturable haemotropic mycoplasma, was estimated using pulsed-field gel electrophoresis (PFGE). Gamma irradiation was used to introduce one (on the average) double-strand break in the E. suis Illinois chromosome. Restriction enzymes that cut infrequently were also used to analyze genome size. The size estimate for the full-length genome was 745 kilobases (kb), whereas the size estimates based on the summation of restriction fragments ranged from 730 to 770 kb. The 16S rRNA gene was located on the 120-kb MluI fragment, 128-kb NruI fragment, 25-kb SacII fragment, and 217-kb SalI fragment by Southern blotting.