Molecular cloning and characterization of murine leukemia virus-related DNA sequences from C3H/HeN mouse DNA.

Ten murine leukemia virus (MuLV)-related DNA sequences were isolated from C3H/HeN mouse genomic DNA by cloning of EcoRI fragments in a Charon 4A vector. Detailed restriction endonuclease maps of four of the clones were developed by using AKR MuLV [32P]cDNA as a probe. C3H clone 14-9 contains approximately 7 kilobase pairs of MuLV-related DNA, one copy of an MuLV long terminal repeat-like sequence, and a region of flanking mouse DNA. C3H clones 34.2 and 36.1 contain approximately 2 kilobase pairs of MuLV-related DNA, one copy of a MuLV LTR-like sequence, and differing lengths of flanking mouse DNA sequences. C3H clone 8.13 was found to contain an insert of 5.7 kilobase pairs of MuLV-related DNA with two long terminal repeat-like regions and sequences which are partially homologous to AKv-1. Comparison fo the restriction endonuclease cleavage maps of these C3H clones with maps recently developed for ecotropic and xenotropic MuLV DNAs indicates that C3H clone 14-9 corresponds to the 5'-terminal portion of a genomic DNA sequence related to xenotropic MuLVs, whereas C3H clones 34.2 and 36.1 correspond to the 3' terminal portions of genomic DNA sequences related to xenotropic MuLVs. Clone 8.13 represents a deleted, xenotropic MuLV-related provirus. C3H clones 14-9, 34.2, 36.1, and 8.13 provide defined DNA sequence probes with which to characterize the organization and expression of endogenous MuLV-related DNA sequences in the mouse genome.     

Isolation and characterization of a sea urchin hsp 70 gene segment

Three clones containing Paracentrotus lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo.     

Isolation and characterization of human actin genes cloned in phage lambda vectors

Using Drosophila and chicken actin probes, we have selected 14 human actin lambda recombinants from a genomic library. We present a restriction maps indicating the positions of the sequences homologous to actin and to an Alu probe. Restriction mapping has revealed that nine out of ten of these clones are distinct, indicating that actin is a multigene family. Hybrid elution of HeLa cell mRNA from filters containing the recombinant DNA, followed by in vitro translation and immunoprecipitation, as well as one- or two-dimensional protein analysis, shows that these recombinants code for actin. Hybridization back to human DNA digested with restriction enzymes shows that the EcoRI fragments of at least one of the lambda recombinants (lambda HA-5) result in similar-sized human DNA fragments in the intact genome. In nuclei, a 4.5-kb mRNA precursor to the cytoplasmic 1.9-kb mRNA can be detected by hybridization with genomic or cDNA probes, indicating the presence of additional sequences and RNA processing.     

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.     

Human cytoplasmic actin proteins are encoded by a multigene family

We characterized nine human actin genes that we isolated (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981) from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and alpha-, beta-, and gamma-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria we show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken beta-actin cDNA. We conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.     

Isolation and characterization of beta- and gamma-crystallin genes from rat genomic cosmid libraries

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.     

Molecular cloning of nif DNA from Azotobacter vinelandii.

Two clones which contained nif DNA were isolated from a clone bank of total EcoRI-digested Azotobacter vinelandii DNA. The clones carrying the recombinant plasmids were identified by use of the 32P-labeled 6.2-kilobase (kb) nif insert from pSA30 (which contains the Klebsiella pneumoniae nifK, nifD, and nifH genes) as a hybridization probe. Hybridization analysis with fragments derived from the nif insert of pSA30 showed that the 2.6-kb insert from one of the plasmids (pLB1) contains nifK whereas the 1.4-kb insert from the other plasmid (pLB3) contains nifD. Marker rescue tests using genetic transformation indicated that the 2.6-kb A. vinelandii nif fragment contains the wild-type alleles for the nif-6 and nif-38 mutations carried by Nif- strains UW6 and UW38. The 1.4-kb insert contains the wild-type allele for the nif-10 mutation carried by Nif- strain UW10.     

The chromosomal arrangement of two linked actin genes in the sea urchin S. purpuratus.

Four distinct actin genes of the sea urchin Strongylocentrotus purpuratus have been isolated from a recombinant Charon 4 phage library of genomic DNA. The four genes differ considerably from each other in many of their restriction sites. Two of the four genes are closely linked; they are present in the same fragment of cloned DNA. This fragment has been extensively mapped, and some parts of the DNA have been sequenced. The two linked genes are oriented in the same direction, separated by 7.5 kb of DNA. One has an intron following the CAG that codes for the glutamine residue at position 121 in the amino acid sequence of actin. This represents the fifth distinct site at which introns have been found in actin genes, suggesting that the primordial actin gene had at least 6 exons and 5 introns. The actin genes from a distinctive family in which most introns have apparently been precisely excised from the genes.     

Linkage arrangement of human placental lactogen and growth hormone genes

Human placental lactogen (hPL) and growth hormone (hGH) are two hormones thought to have evolved from a common ancestral gene (along with prolactin), yet they have quite different functions and specificities. The nucleic acid sequences of the respective cDNAs of the two genes share considerable homology, as well as the existence of multiple forms of each gene within the genome. In this study we report on the linkage arrangement of several genes from this group. Two hPL-like genes as well as an hGH gene are shown to be linked within a 38-kilobase pair region of DNA. Linkage between a variant hGH gene and an hPL gene is also shown. The orientation and structural organization of these genes was previously established using 5'- and 3'-specific probes from a placental lactogen cDNA clone and detailed restriction endonuclease mapping. Restriction fragments from the overlapping clones were verified by comparison to digests of high molecular weight genomic DNA. In addition, the location of a specific class of repetitive DNA sequences, the Alu family, was mapped on these clones using the recombinant clone BLUR 8. All members of this multigene family have Alu repeat sequences either immediately flanking their 3' or 5' untranslated regions or within their intervening sequences.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

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.     

Sea urchin actin gene subtypes. Gene number, linkage and evolution

The actin gene family of the sea urchin Strongylocentrotus purpuratus was analyzed by the genome blot method, using subcloned probes specific to the 3' terminal non-translated actin gene sequence, intervening sequence and coding region probes. We define an actin gene subtype as that gene or set of genes displaying homology with a given 3' terminal sequence probe, when hybridized at 55 degrees C, 0.75 M-Na+. By determining the often polymorphic restriction fragment band pattern displayed in genome blots by each probe, all, or almost all of the actin genes in this species could be classified. Our evidence shows that the S. purpuratus genome probably contains seven to eight actin genes, and these can be assigned to four subtypes. Studies of the expression of the genes (Shott et al., 1983) show that the actin genes of three of these subtypes code for cytoskeletal actins (Cy), while the fourth gives rise to a muscle-specific actin (M). We denote the array of S. purpuratus actin genes indicated by our data as follows. There is a single CyI actin gene, two or possibly three CyII genes (CyIIa, CyIIb, and possibly CyIIc), three CyIII actin genes (CyIIIa, CyIIIb, CyIIIc), and a single M actin gene. Comparative studies were carried out on the actin gene families of five other sea urchin species. At least the CyIIa and CyIIb genes are also linked in the Strongylocentrotus franciscanus genome, and this species also has a CyI gene, an M actin gene and at least two CyIII actin genes. It is not clear whether it also possesses a CyIIc actin gene, or a CyIIIc actin gene. The genome of a more closely related congener, Strongylocentrotus dr?bachiensis, includes 3' terminal sequences suggesting the presence of a CyIIc gene. In S. franciscanus and S. dr?bachiensis the first intron of the CyI gene has remained homologous with intron sequences of both the CyIIa and CyIIb genes, indicating a common origin of these three linked cytoskeletal actin genes. Of the four S. purpuratus 3' terminal subtype probe sequences only the CyI 3' terminal sequence has been conserved sufficiently during evolution to permit detection outside of the genus Strongylocentrotus. An unexpected observation was that a sequence found only in the 3' untranslated region of the CyII actin gene in the DNA of S. dr?bachiensis and S. purpuratus is represented as a large family of interspersed repeat sequences in the genome of S. franciscanus.     

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.