The effect of 1-deoxymannojirimycin on rat liver alpha-mannosidases

Cloned murine mammary tumor virus (MuMTV) sequences allowed us to search for murine mammary tumor virus related sequences in the DNA of surgically removed human breast tumors. Out of 28 tumors so far examined two were found to contain an Eco RI DNA fragment homologous to the long terminal repeat-group antigen (LTR-Gag) and the Envelope (Env) sequences of MuMTV. We have taken the lymphocytes of these patients and cultured them. Rapid growth of lymphocytes, mostly of T origin, occurred in the presence of T cell growth factor (TCGF). Whereas DNA extracted from fresh lymphocytes is negative, that extracted from the 3-day cultured lymphocytes showed MuMTV related sequences. Long term cultures of T cells and a similar culture derived from a healthy person donor were negative at all stages. DNA extracted from the Ebstein Barr Virus-transformed B cells of the patient does not contain the MuMTV related sequences.     

Integration of New Endogenous Mouse Mammary Tumor Virus Proviral DNA at Common Sites in the DNA of Mammary Tumors of C3Hf Mice and Hypomethylation of the Endogenous Mouse Mammary Tumor Virus Proviral DNA in C3Hf Mammary Tumors and Spleens

To understand the molecular mechanisms by which the endogenous murine mammary tumor virus (MuMTV) proviruses are expressed and produce late-occurring mammary tumors in C3Hf mice, we analyzed, by the use of restriction enzymes and the Southern transfer procedure, genomic DNA from normal organs of mammary tumor-bearing and tumor-free mice and from 12 late-occurring C3Hf mammary tumors. We found, by using the restriction enzymes EcoRI and HindIII, that in addition to the preexisting endogenous MuMTV proviruses, new MuMTV-specific proviral DNA was integrated into new sites in the host genome in all 12 of the tumors that we examined. PstI digests of C3Hf tumor DNA revealed that the new proviral DNA found in C3Hf tumors was of endogenous origin. Moreover, the respective sizes of at least one of the new DNA fragments generated by EcoRI or HindIII digestion were the same in at least 50% of the C3Hf tumors analyzed, suggesting that the integration site of this new proviral DNA could be at the same location in the host genome of these tumors. Our results may imply that mammary tumorigenesis in C3Hf mice results from activation of cellular oncogenes by an MuMTV proviral DNA promoter. Specific hypomethylation of MuMTV proviral DNA was detected in the mammary tumors and spleens of C3Hf tumor-bearing mice. Our results indicated that most, if not all, of the hypomethylated MuMTV proviral DNA sequences were derived from the endogenous MuMTV provirus located at the MTV-1 locus, a locus responsible for the production of MuMTV antigens and increased incidence of mammary carcinoma in C3Hf mice. In spleens of non-tumor-bearing mice of ages 3, 6, 9, and 12 months, there was progressive hypomethylation of proviral DNA with increasing age, suggesting a possible correlation between demethylation of MuMTV proviral DNA in the spleens of C3Hf mice and the expression of endogenous MuMTV.     

The human genome contains multiple sequences of varying homology to mouse mammary tumour virus DNA

Sequences related to the mouse mammary tumour virus (MuMTV) DNA were isolated from a genomic library of human DNA by screening under conditions of relaxed stringency. It is estimated that there are in the order of 50 MuMTV-like sequences per haploid genome and that the homology between the different human sequences and MuMTV varies by 15%.     

Presence of antibodies specifically reacting with structural proteins of the mammary cancer virus among antibodies found in circulating immune complexes in patients with breast cancer

Circulating immune complexes were precipitated from breast cancer patients' sera using 2.5% polyethylene glycol. CIC isolated from 70 ml of sera from 15 patients were dissociated and immunoglobulin-containing fraction was prepared by chromatography on Sephadex G-200 column. The fraction contained IgG specific for MuMTV structural proteins, as revealed by ELISA. CIC preparations from 22 sera of breast cancer patients were digested with pepsin; Fab' fragment preparations were also analysed by ELISA, only one of them was MuMTV-specific. IgG and Fab' fragments isolated from CIC reacted specifically with MuMTV proteins, the reaction was not blocked by virus-free murine milk or other retroviruses (Ra-MuLV and MPMV).     

Autonomous replicating sequences from intron of human ras gene in a simian virus 40 T antigen dependent system

Of the several DNA fragments present in the human lung cancer gene, 1.1 and 2.0 kilobase (kb) fragments corresponding to the intron of this gene were hybridized to a half part of the 27 nucleotides perfect palindrome present in the initiation part of replication in simian virus 40 (SV40) DNA. These two fragments cloned in pBR322 had good template activity, and the initiation of DNA replication started from the region of these fragments in an in vitro system, in which the initiation of DNA replication occurs on cloned DNA containing SV40 origin of DNA replication as described previously. Furthermore, these two clones could replicate autonomously in nuclei of SV40 transformed Cos cells, producing SV40 T antigen constitutively when the clones were transfected into Cos cells. These results show that functional SV40 origin-like sequences are present in human genomes, and they can replicate autonomously within the cells which are producing SV40 T antigen.     

Identification of novel breast tumor-specific mutation(s) in the q11.2 region of chromosome 17 by RAPD/AP-PCR fingerprinting

Analysis of genetic instability in breast cancer tissues compared to uninvolved breast tissues from the same individuals by RAPD (random amplified polymorphic DNA)/AP-PCR (arbitrarily primed PCR) fingerprinting using 30 arbitrary primers revealed 190 amplified DNA fragments. Presumably, each of these represents a gene locus in a different region of the genome of breast cancer tissues. Among these amplified DNA fragments, 65 (34.2%) exhibited presence and absence or reductions and enhancements in the intensity in breast cancer tissues compared to uninvolved breast tissues from the same individuals, and 11 amplified DNA fragments (5.7%) represented polymorphisms in the uninvolved human breast tissues. Reductions and enhancements in the intensity of some of the amplified fragments were observed indicating allelic gains or losses in the breast tumor genome compared to the matched uninvolved tissue genome. The presence or absence of some of the amplified DNA fragments were observed in this study indicating homozygous deletions or insertions in the breast tumor DNA compared to the matched uninvolved tissue DNA. Notably, an insertion of a 1270 bp amplified fragment was observed in 81% (17 of 21) of the tumor samples using the primer, OPC04. This amplified fragment resolved into two, 1200 and 1300 bp, single-stranded amplified fragments on the denaturing sequencing gel. This separation into single-stranded fragments suggests that the amplified fragment contains a conformation that is semistable. The 1270 bp amplified fragment localizes to the q11.2 region of chromosome 17. Sequence analysis of this fragment showed a significant DNA base sequence similarity (93%) with one of the breast tumor-specific human EST. The similarity with EST sequences and RT-PCR analysis showed that a part of this amplified fragment is from the coding region of the genome. Any one of the events observed in this study could play an important role in the development of breast cancer or could occur during the clonal expansion of the genetically unstable breast cells.     

Human thymidylate synthase gene: isolation of phage clones which cover a functionally active gene and structural analysis of the region upstream from the translation initiation codon

Two genomic DNA fragments partially encoding human thymidylate synthase (TS) [EC 2.1.1.45] were previously cloned in lambda phage from the mouse cell transformant, but had no transforming activity on mouse TS-negative mutant cells. In this study, an additional genomic DNA for human TS was cloned and demonstrated to have the transforming activity in combination with one of the two previously cloned DNAs and to produce human TS mRNA. The two transforming genomic DNAs overlapped and covered a region of 23 kb in total. Using fragments from one of these DNAs, the structure of the 1.2-kb region around the ATG initiator codon of the TS gene was analyzed in relation to regulatory sequences of the gene. Sequence determination demonstrated the presence of an unusual inverted repeat consisting of a triple tandem repeat of a 28-bp sequence and an inverted sequence of the same length. These sequences can form three possible, stable, stem-loop structures, which may be interconvertible. Based on S1 nuclease mapping data and a line of circumstantial evidence, we deduced two major mRNA cap sites within the inverted sequence. Comparison of the human and mouse sequences upstream from the ATG initiator codon revealed many significant blocks of sequence homology, especially in the regions around the deduced cap sites.     

Characterization of a highly repetitive family of DNA sequences in the mouse.

A large proportion (0.5-1%) of total mouse DNA is cleaved by Bam HI into fragments whose size is about 500 base pairs. A cloned member of this repetitive family of DNA sequences (BAM5 family) was sequenced by the dideoxy chain termination procedure and shown to contain 507 base pairs. The sequence exhibited no unusual or remarkable features. Repetitive sequences complementary to the cloned BAM5 fragment were found in rat DNA, but not in feline or human DNA. Restriction mapping suggested that many BAM5 sequences were components of much larger repetitive DNAs which were scattered throughout the mouse genome. The BAM5 sequences within the larger repetitive DNAs did not appear to be arranged tandemly or as members of scrambled tandem repeats. RNA homologous to the cloned BAM5 sequence was detected in cultured mouse cells, but not in cultured rat cells.     

Purification and characterization of a complex containing matriptase and a Kunitz-type serine protease inhibitor from human milk.

Matriptase, a trypsin-like serine protease with two potential regulatory modules (low density lipoprotein receptor and complement C1r/s domains), was initially purified from T-47D breast cancer cells. Given its plasma membrane localization, extracellular matrix-degrading activity, and expression by breast cancer cells, this protease may be involved in multiple aspects of breast tumor progression, including cancer invasion. In breast cancer cells, matriptase was detected mainly as an uncomplexed form; however, low levels of matriptase were detected in complexes. In striking contrast, only the complexed matriptase was detected in human milk. The complexed matriptase has now been purified. Amino acid sequences obtained from the matriptase-associated proteins reveal that they are fragments of a Kunitz-type serine protease inhibitor that was previously reported to be an inhibitor of the hepatocyte growth factor activator. In addition, matriptase and its complexes were detected in milk-derived, SV40 T-antigen-immortalized mammary luminal epithelial cell lines, but not in human foreskin fibroblasts or in HT-1080 fibrosarcoma cells. These results suggest that the milk-derived matriptase complexes are likely to be produced by the epithelial components of the lactating mammary gland in vivo and that the activity and function of matriptase may be differentially regulated by its cognate inhibitor, comparing breast cancer with the lactating mammary gland.     

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.     

Cloning of V region fragments from mouse liver DNA and localization of repetitive DNA sequences in the vicinity of immunoglobulin gene segments.

Two different kappa light chain genes have previously been isolated from one mouse myeloma. The V (variable, abbreviations in ref. 2) gene segments of the two genes were now used to identify their germline counterparts in EcoRI digests of mouse liver DNA. In addition two sets of related V gene segments were found which hybridize with either of the two DNA probes. Five of the V region fragments of one set were cloned in a lambda phage vector and partially characterized by restriction mapping and Southern blot hybridization. Repetitive DNA sequences were found on each of the five fragments as well as on other cloned immunoglobulin gene containing fragments. Cross-hybridization between some but not all of the regions containing repetitive DNA sequences was observed.     

Murine mammary tumor virus: characterization of infection of nonmurine cells.

Murine mammary tumor virus (MuMTV) was used to productively infect feline and mink cells. MuMTV "proviral" DNA could be detected in the infected cells by molecular hybridization using radioactive MuMTV complementary DNA as a probe. Kinetic analysis of MuMTV proviral DNA synthesis after infection showed that maximum MuMTV DNA synthesis was achieved by 8 h; however, this was followed by a decline in detectable proviral DNA and eventual stabilization at a lower level. MuMTV synthesis in feline cells was greatly stimulated by the synthetic glucocorticoid, dexamehtasone. On the other hand, MuMTV synthesis in mink cells was relatively at a much higher level in absence of dexamethasone and the stimulation with dexamethasone was not as marked as in the case with infected feline cells. Thermal denaturation of hybrids between MuMTV complementary DNA and infected mink cell RNA revealed no difference from homologous hybrids.     

Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments

Restriction enzyme XbaI DNA fragments that represent 99% of the sequences from the long and short unique as well as the repeat sequences of the human cytomegalovirus (CMV) genome have been cloned into bacterial plasmid pACYC184. The viral DNA sequences associated with the recombinant plasmids were analyzed by restriction mapping and by hybridization to fragments of authentic viral DNA. The relationship of the cloned viral DNA fragments to the XbaI physical map of the viral genome is demonstrated. Even though large recombinant plasmids ranging from approx. 39 to 1.8 kb were isolated, most if not all of the viral DNA fragments were stable during propagation in Escherichia coli HB101.     

The retrieval of ancient human DNA sequences.

DNA was extracted from approximately 600-year-old human remains found at an archaeological site in the southwestern United States, and mtDNA fragments were amplified by PCR. When these fragments were sequenced directly, multiple sequences seemed to be present. From three representative individuals, DNA fragments of different lengths were quantified and short overlapping amplification products cloned. When amplifications started from <40 molecules, clones contained several different sequences. In contrast, when they were initiated by a few thousand molecules, unambiguous and reproducible results were achieved. These results show that more experimental work than is often applied is necessary to ensure that DNA sequences amplified from ancient human remains are authentic. In particular, quantitation of the numbers of amplifiable molecules is a useful tool to determine the role of contaminating contemporary molecules and PCR errors in amplifications from ancient DNA.