Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.     

Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

Isolation and characterization of genomic DNA coding for alpha 2 type I collagen.

We have isolated and characterized a segment of the chick alpha 2 collagen gene by screening a library of chick genomic fragments using as hybridization probe an alpha 2 collagen cDNA clone. Several clones were isolated and one of them, lambda gCOL 204, was used for further studies. The DNA of lambda gCOL 204 hybridizes to a unique species of mRNA the size of alpha 2 collagen mRNA. This mRNA can be translated into a unique polypeptide which comigrates in SDS-gel electrophoresis with pro-alpha 2 collagen. Electron microscopic analysis by R-loop technique indicates that lambda gCOL 204 contains 7Kb of the alpha 2 collagen gene. This 7 Kb piece constitutes the 3' end of the gene. The same clone also contains 9 Kb of DNA that is immediately adjacent to the 3' end of the alpha 2 collagen gene. The cloned segment of the alpha 2 collagen gene is interrupted by 8 intervening sequences of various lengths. The coding sequences for collagen in this clone add up to approximately 1,800 bp, which correspond to about 1/3 of alpha 2 collagen mRNA. DNA sequence analysis of a small coding segment of lambda g COL 204 reveals a characteristic collagen type sequence which encodes for an amino acid sequence identical to a sequence found in calf alpha 2 collagen. The sequence of this region of the protein has not yet been determined for the chick alpha 2 collagen.     

Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.     

Allotypes of the constant region of the rabbit T cell receptor beta-2 chain

Our laboratory previously reported that there was restriction fragment length polymorphism of TCR C beta genes in rabbits. EcoRI digests of DNA from different rabbits gave fragments of 9 and 6 kb (C beta a) or 14 and 6 kb (C beta b) that hybridized to a C beta cDNA probe. We also reported that the 9- and 14-kb types segregated as Mendelian traits and that there were allotypic differences in the first exon of the C beta 1 genes of C beta a and C beta b animals. Here we report the DNA sequence of the C beta 2 gene present in the 6-kb EcoRI fragment from a C beta b animal and compare the exon sequences with that of a cDNA from a C beta a animal. We find replacement changes in the first and third exons that probably represent allotypic forms of the rabbit C beta 2 gene. The genomic DNA 5' of exon 1 of both beta 1 and beta 2 contain alternating purine/pyrimidine repeat sequences. The genomic C beta 2 has an open reading frame of 69 amino acids in frame with exon 1 similar to a longer one previously found 5' of exon 1 of C beta 1. Further 5' of this region, rabbit C beta 1 and C beta 2 DNA sequences are only about 66% similar. Both the C beta 1 and C beta 2 sequences have two chi sequences; one in exon 1 with a perfect match and one in the intron downstream of exon 1 with one mismatch. Alternating purine/pyrimidine repeats and chi sequences found in rabbit C beta 1 and C beta 2 genes may have contributed to process(es) of gene duplication and/or conversion.     

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.     

Variation between mouse major urinary protein genes isolated from a single inbred line

We describe ten Charon 4A genomic DNA clones from BALB/c mice which include at least seven different major urinary protein (MUP) genes. We have established the orientation of all seven sequences, and have placed six of them in precise register by means of restriction site maps and Southern blot hybridization with cloned cDNA sequences. Four of the seven genomic sequences (family I sequences) form hybrids with six independent cDNA clones that have a high thermal stability and hybridize more strongly with mRNA from three inbred mouse lines. Hybrids between the remaining three genomic sequences and the cDNA clones have a lower thermal stability and hybridize less strongly with mRNA from the three inbred lines. Homologies between different cloned sequences extend over as much as 15 kb. No clone contains parts of two MUP genes, and no homology has been detected between the 3' flanking region of one MUP gene and the 5' flanking region of another.     

Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase.

A fragment of DNA from the yeast nuclear gene MST1 that codes for the mitochondrial tRNAThr1 synthetase was used as a probe to screen for other yeast threonyl-tRNA synthetase genes. At low stringency, the MST1 probe hybridizes strongly to a 6.6 kb EcoRI fragment of yeast genomic DNA with the homologous gene and in addition hybridizes more weakly to a smaller 3.6 kb EcoRI fragment with a second threonyl-tRNA synthetase gene (THS1). To clone THS1, a library was constructed by ligation to pUC18 of size selected (3-4.5 kb) EcoRI fragments of genomic DNA. Several clones containing the 3.6 kb EcoRI fragment were isolated. A 2,202 nucleotide long open reading frame corresponding to THS1 has been identified in the cloned fragment of DNA. The predicted protein encoded by THS1 is 38% identical to the E. coli threonyl-tRNA synthetase over the latter's length (642 amino acids) and is 42% identical to the predicted MST1 product over its 462 residues. In situ disruption of the chromosomal copy of THS1 is lethal to the cell, indicating that this gene codes for the cytoplasmic threonyl-tRNA synthetase.     

Cloning and nucleotide sequence of the murine interleukin-3 gene

Southern hybridization analysis using a probe derived from a murine interleukin-3 (IL-3) cDNA clone revealed the presence of a single IL-3 gene in the haploid murine genome. An 8600-base-pair (8.6-kb) murine genomic EcoRI fragment containing the IL-3 gene was isolated by screening a library of size-fractionated genomic EcoRI fragments cloned in lambda gtWES X lambda B. The nucleotide sequence of a 3.5-kb region of the cloned DNA encompassing the IL-3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14-15-bp tandem repeating sequence which resembles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL-3 cDNA cloned from WEHI-3, a tumorigenic cell line which over-produces IL-3, establishing that the unprocessed primary structure of IL-3 is identical in WEHI-3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL-3 gene in WEHI-3 cells.     

A rearranged DNA sequence possibly related to the translocation of immunoglobulin gene segments.

A 5.3 kb EcoRI fragment (T3, abbreviations in ref. 2) has been cloned from DNA of a kappa light chain producing mouse myeloma. The fragment hybridizes to the k' flanking sequences of the J1 gene segment but not to C gene sequences of kappa light chain DNA. Restriction nuclease mapping and partial nucleotide sequencing showed that the fragment consists of sequences from the 5' side of the J1 and form the 3' side of a V gene segment, which apparently had been linked in a genomic rearrangement process. These rearranged flanking sequences are not the flanking sequences of the V and J gene segments which had been joined to form the two kappa light chain genes of the myeloma. Fragments with the hybridization properties of T3 have been found also in two other kappa and one lambda chain producing myelomas. The linking of flanking sequences in the myeloma genome is discussed with respect to the mechanism of recombination between V and J gene segments.     

Identification of four genomic loci highly related to casein-kinase-2-alpha cDNA and characterization of a casein kinase-2-alpha pseudogene within the mouse genome.

Using the coding region of the human CK-2 alpha cDNA as a probe for screening a genomic mouse library, positive clones representing four different genomic loci were isolated. Partial DNA sequences of these loci encompassing the first 120 nucleotides of the putative coding region are reported. One positive clone was further analyzed by sequencing a 3.1 kb XbaI fragment. This clone displays the characteristics of a pseudogene, i.e. lack of introns and several nucleotide insertions and deletions. In its 3' region it contains a 91 bp large CT-rich stretch which consists of (CCTT) and (CT) repeats; in the 5' region three (CCCCCT) repeats.     

Isolation and characterization of the catalase gene from Rhizobium sp. SNU003, a root nodule symbiont of Canavalia lineata

A catalase gene from Rhizobium sp. SNU003, a root nodule symbiont of Canavalia lineata, was cloned and its nucleotide sequence was determined. The Rhizobium DNA of about 280 bp was amplified using two PCR primers synthesized from the conserved sequences of the type I catalase gene. The nucleotide sequence of the amplified fragment revealed three regions that were conserved in the catalase, showing it as being part of the catalase gene. A genomic Southern hybridization using this fragment as a probe showed that the 5.5 kb PstI, 1.8 kb EcoRI, and 0.7 kb StyI fragments hybridized strongly with the probe. The Rhizobium genomic library constructed into the EMBL3 vector was screened, and one catalase clone was selected. The nucleotide sequence of the 5.5 kb PstI fragment from the clone revealed an open reading frame of 1455 bp, encoding a polypeptide of 485 amino acids with a molecular mass of 54,958 Da and a pI of 6.54. The predicted amino acid sequence of the catalase is 66.3% identical to that of Bacteroides fragilis, but was only 53.3% identical to the Rhizobium meliloti catalase.