Location of DNA sequences complementary to small nuclear repeat RNA (fr 3-RNA) in relation to DNA sequences coding for albumin and alpha-fetoprotein in rat liver cells

Rat liver nuclei contain a 29-nucleotides-long RNA (fr 3-RNA) which is transcribed from middle repetitive DNA sequences. By Southern analysis of restriction fragments of rat albumin and alpha-fetoprotein genomic clones, DNA sequences complementary to this RNA were detected on a 4.6 kbp Eco RI fragment located 600 bp downstream from the termination exon of the albumin gene and on a 2 kbp Eco RI-HindIII fragment located 10 kbp downstream from the restriction fragment containing the alpha-fetoprotein site. No sequence complementary to this RNA was found either in the introns of exons of both genes or in the regions extending 7 kbp upstream from the first albumin exon and 10 kbp upstream of the first alpha-fetoprotein exon. We concluded that sequences complementary to fr 3-RNA are present at the 3'-end flanking regions of the rat albumin and alpha-fetoprotein gene complexes.     

Location of DNA sequences complementary to small nuclear repeat RNA (fr 3-RNA) in relation to DNA sequences coding for albumin and α-fetoprotein in rat liver cells

Rat liver nuclei contain a 29-nucleotides-long RNA (fr 3-RNA) which is transcribed from middle repetitive DNA sequences. By Southern analysis of restriction fragments of rat albumin and α-fetoprotein genomic clones, DNA sequences complementary to this RNA were detected on a 4.6 kbp EcoRI fragment located 600 bp downstream from the termination exon of the albumin gene and on a 2 kbp EcoRI-HindIII fragment located 10 kbp downstream from the restriction fragment containing the α-fetoprotein site. No sequence complementary to this RNA was found either in the introns of exons of both genes or in the regions extending 7 kbp upstream from the first albumin exon and 10 kbp upstream of the first α-fetoprotein exon. We concluded that sequences complementary to fr 3-RNA are present at the 3′-end flanking regions of the rat albumin and α-fetoprotein gene complexes.     

Use of the mouse mammary tumor virus long terminal repeat to promote steroid-inducible expression of v-mos.

We used the mouse mammary tumor virus long terminal repeat to promote dexamethasone-regulated expression of the Moloney murine sarcoma virus (M-MSV) transforming gene, v-mos. A recombinant DNA vector containing the mouse mammary tumor virus long terminal repeat fused to the M-MSV 124 v-mos gene was cotransfected with a plasmid containing the herpes simplex virus thymidine kinase gene (tk) into 3T3TK- cells. Individual clones of cells which grew in hypoxanthine-aminopterin-thymidine medium were tested for dexamethasone-regulated expression of p37mos as well as several transformation-specific phenotypic parameters. In the absence of dexamethasone, the v-mos transfectants appeared morphologically similar to the control cells despite low basal levels of p37mos expression. Upon hormone treatment, the levels of p37mos increased 5- to 10-fold, coincident with morphological changes typical of M-MSV transformation of 3T3 cells. The ability to form foci in monolayers also correlated with p37mos induction. The extent of morphological changes varied in individual clones of cells with similar levels of induced p37mos. Although the induced levels of p37mos were comparable to those seen in stable M-MSV 124 virus-transformed NIH 3T3 cells, the transfectants were unable to grow in soft agar under conditions which support growth of the virus-transformed cells. Acute infection of the transfectants with M-MSV 124 virus, a situation which resulted in elevated levels of p37mos, allowed these cells to grow in soft agar. The results described in this paper suggest that different threshold levels of p37mos may be necessary for the expression of various parameters of the transformed phenotype and also that continued expression of p37mos is necessary for maintenance of the transformed state. However, it also appears that the sensitivity to given levels of p37mos varies among clonal cell lines.     

Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus.

BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.     

Cloning and dosage compensation of the 6-phosphogluconate dehydrogenase gene (Pgd+) of Drosophila melanogaster

Using a heterologous rat cDNA probe, we have identified a 14.7 kbp Drosophila melanogaster genomic clone containing the X-linked gene Pgd+, which encodes the enzyme 6-phosphogluconate dehydrogenase (6PGD). We used in situ hybridization to larval polytene chromosomes, a somatic transient expression assay for enzyme activity, and the rescue of the lethal Pgd- phenotype by germline transformation to verify the identity of the gene. A 7.4 kbp fragment including the gene and approximately 1.2 kbp of upstream and 1.8 kbp of downstream sequences was relocated to autosomal ectopic sites by germline transformation; this transduced gene exhibits levels of enhanced activity in males comparable to those of the indigenous gene at its normal X chromosome locus. We conclude that the sequences responsible for dosage compensation of Pgd+ are included in this fragment.     

The basidiomycete Lentinus edodes linear mitochondrial DNA plasmid contains a segment exhibiting a high autonomously replicating sequence activity in Saccharomyces cerevisiae.

A linear DNA plasmid, designated pLLE1, has been isolated from a mitochondrial fraction of a strain of Lentinus edodes. pLLE1(11.0 kbp) was sensitive to the 3'----5'-acting exonuclease III and resistant to the 5'----3'-acting lambda exonuclease. It showed no homology with mitochondrial and nuclear genomic DNAs of plasmidless strain as well as the pLLE1-harboring host strain of L. edodes. The 1434-bp fragment (sequences) capable of autonomous replication in the yeast Saccharomyces cerevisiae (ARSs) was cloned from pLLE1 DNA with YIp32 (pBR322 containing yeast LEU2 DNA), which displayed a high ARS activity. The cloned 1434-bp fragment was shown to lie near to the end of pLLE1 DNA (nucleotides about 800-2200) and contained three consecutive ARS consensus sequences (A/T)TTTAT(A/G)TTT(A/T) of S. cerevisiae and dispersive eight ARS consensus-like sequences. The subcloned 366-bp fragment containing the three ARSs retained original ARS activity of the 1434-bp fragment.     

Transforming genes in human tumors

DNAs isolated from a variety of human tumor cell lines as well as from naturally occurring human carcinomas and sarcomas were shown to induce morphologic transformation upon transfection into NIH/3T3 cells. All tested transformants contained human DNA sequences, some of which specifically cosegregated with the malignant phenotype in additional cycles of transfection. Southern blot analysis of second cycle transformants derived from T24 human bladder carcinoma cells showed the presence of a single 15 kbp EcoRI fragment of human DNA. These sequences were molecularly cloned utilizing λ Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated λ T24-15A, was shown to contain an internal 6.6 kbp Bam HI fragment of human DNA that transformed NIH/3T3 fibroblasts with a specific activity of 5 × 10⁴ focus forming units per picomol. These results indicate that we have moleculary cloned an oncogene present in T24 bladder carcinoma cells. Comparison of molecular clones containing the T24 oncogene and its normal homologue did not reveal biochemical differences that helped to explain the malignant properties of this oncogene. Finally, we report preliminary results indicating that the T24 bladder carcimoma oncogene is highly related to the transforming gene of BALB-MSV, an acute transforming retrovirus.     

High level expression of human apolipoprotein A-I in transgenic rats raises total serum high density lipoprotein cholesterol and lowers rat apolipoprotein A-I

To examine the consequences of increased apolipoprotein A-I production on cholesterol and lipoprotein metabolism, we have produced two lines of transgenic rats; one expressing moderate and one very high levels of human apolipoprotein A-I. The rats were produced by microinjection of a 13 kbp DNA fragment containing the human apolipoprotein A-I gene plus 10 kbp of its 5′ flanking sequence and 1 kbp of its 3′ flanking sequence. Both lines of transgenic rats express human apolipoprotein A-I mRNA in liver and human apolipoprotein A-I in plasma. Sera from these rats contain significantly higher levels of total apolipoprotein A-I, high density lipoprotein cholesterol and phospholipid than sera from non-transgenic littermates. Transgenic rats expressing high levels of human apolipoprotein A-I have reduced levels of serum rat apolipoprotein A-I suggesting a mechanism exists to down-regulate apolipoprotein A-I production. These transgenic rats provide a unique animal model to examine the effects of increased apolipoprotein A-I production on lipid and lipoprotein metabolism.     

Construction of a NotI linking library and isolation of new markers close to the Huntington''s disease gene.

Linking clones contain sequences flanking recognition sites for enzymes cutting rarely in mammalian DNA. They can be used to obtain and correlate both physical and genetic mapping information over subregions of mammalian chromosomes. We have constructed and used a NotI linking clone library representing unmethylated NotI sites from HHW693 DNA, a hamster hybrid cell line containing 4p15-4pter and a fragment of 5p as its only human chromosome contribution. Human clones were identified by hybridisation with a cloned human repeat sequence, and localised further to subregions of human chromosome 4p15-4pter using a panel of additional hybrids. Clones from the region distal to the DNA probes (D4S10, D4S43, D4S95) linked to the Huntington's disease mutation, were further analysed. Four markers close to the HD gene: D4S111, D4S113, D4S114 and clone 417 are described here. In addition to serving as markers in physical and genetic mapping experiments, these linking clones provide probes next to cleavable NotI sites, and can therefore be used to screen NotI based chromosome jumping libraries. They also provide indications for potential gene sequences, identifiable as evolutionarily conserved sequences.     

Complete c-mos (rat) nucleotide sequence: presence of conserved domains in c-mos proteins

Recently we described the isolation of c-mos (rat). The gene belongs to the family of oncogenes. Some facts render c-mos unique among the oncogenes : a) it does not contain intervening sequences and b) its expression was never detected in a large number of normal mouse tissues examined. We undertook the sequence analysis of c-mos (rat) in order to compare it to the nucleotide sequences published for c-mos (mouse), c-mos (human), c-src and bovine protein kinase. c-mos (rat) contains an open reading frame of 1017 nucleotides, coding for a polypeptide of 339 amino acids. c-mos (rat)-makes use of the same ATG that defines the N-terminus of the c-mos (human) protein. By comparing all c-mos sequences available we found sequences with high mutational rates to be confined to certain domains. This comparison, together with data on the biological activities of the cloned DNA, allowed us to tentatively define regions involved in (a) function(s) of c-mos other than transformation.     

Structural and biological analysis of integrated polyoma virus DNA and its adjacent host sequences cloned from transformed rat cells.

EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA.     

Structural features of the integration site of foreign DNA in the transgenic mouse genome

The structure of the transgenic mouse DNA region containing an integrated transgene (fragment of pBR322 sequence) was analysed. In one of the sequences flanking the transgene, short direct and inverted overlapping repeats were revealed at a distance of 60 bp from the integration site. In the same flanking sequence, there is an extended sequence (3.5 kbp) 0.3-1 kbp away from the transgene. It repeats 100-300 times in the mouse genome and is highly conservative (the homologs of the repeat have been revealed in other mammalian, bird, fish and insect genomes). This up-to-date unknown family of highly-conserved dispersed repeats has been denoted by T1. We believe that both the revealed short inverted repeats capable of forming hairpins with loops and the T1 repeat are structures involved in the process of non-homologous insertion of foreign DNA into the region of the transgenic mouse genome.     

Molecular cloning, characterization, and genetic mapping of an endogenous murine mammary tumor virus proviral unit I of C3H/He mice.

We have cloned and characterized a novel endogenous murine mammary tumor virus proviral unit of the C3H/He strain of mice. The cloned proviral unit is 16 kilobase pairs (kbp) in size and is composed of a 5.6-kbp 5' EcoRI segment of an endogenous provirus with 10.4-kbp flanking cellular sequences. A comparison of the restriction map of the cloned proviral DNA with that of an endogenous provirus of the GR strain of mice has revealed minor differences in restriction sites on the two proviruses. The restriction enzyme SstI, which does not cleave the 5' EcoRI fragment of GR DNA, cleaves the C3H/He proviral sequences once; MspI has an additional site in the C3H/He proviral sequences. By using a subcloned fragment containing unique cellular sequences as a hybridization probe, we (i) mapped the C3H/He proviral unit to chromosome 14 by using mouse-hamster somatic cell hybrids, and (ii) demonstrated that this proviral unit is also present in the genome of DBA/2 mice. From these results we conclude that the C3H/He strain of mice acquired this proviral unit from DBA stock by genetic transmission. Our data also indicate that the murine mammary tumor virus sequences present in the gag-specific proviral unit of C3H/He mice extend at least 2.45 kbp downstream of the EcoRI site in the genomic DNA. Since the structural organization and chromosomal location of this proviral unit are distinct from those of previously reported proviral units represented by similar-sized (16.7-kbp) EcoRI fragments, we tentatively propose to designate this proviral unit Mtv-7a.     

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.