Multiple point mutations affecting the simian virus 40 enhancer.

Enhancers, or activators, dramatically increase the transcriptional activity of certain eukaryotic genes. A series of multiple point mutations affecting the simian virus 40 (SV40) enhancer-activator region were generated in order to define the nucleotide sequence required for this function. Three independent assays provided information leading to the identification of nucleotides essential for enhancer function. One class leads to a decrease in gene expression, while the second completely abolishes functional activity. One critical replacement appears to be the first G (guanine) in a sequence TGGAAAG (T, thymine, A, adenine) located in the 5' region of the 72 base-pair repeat of SV40. Comparison of this sequence with nucleotide sequences in other known enhancers leads to the identification of potential related core elements.

Bactericidal activity of daptomycin, vancomycin, teicoplanin and linezolid against Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium using human peak free serum drug concentrations.

The bactericidal activities of daptomycin, vancomycin, teicoplanin and linezolid at human peak free serum concentrations (C(max,free)) were determined against Staphylococcus aureus (one methicillin-susceptible and two methicillin-resistant strains), Enterococcus faecalis and Enterococcus faecium (one vancomycin-susceptible and one vancomycin-resistant strain of each). Daptomycin was rapidly bactericidal against 7/7 strains at C(max,free) of 22.0 mg/L (corresponding to 63% protein binding) and against 3/7 strains at 4.8 mg/L (corresponding to 92% protein binding). Vancomycin (18.0 mg/L) was bactericidal against only two strains. Both teicoplanin (4.5 mg/L) and linezolid (10.4 mg/L) were consistently bacteriostatic. Daptomycin is a useful option for the treatment of Gram-positive infections owing to its strong bactericidal activity.

Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit.

Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events.

Proviral inactivation of the Npat gene of Mpv 20 mice results in early embryonic arrest.

The Mpv 20 transgenic mouse strain was created by infection of embryos with a defective retrovirus. When Mpv 20 heterozygous animals were crossed, no homozygous neonatal mice or midgestation embryos were identified. When embryos from heterozygous crosses were cultured in vitro, approximately one quarter arrested as uncompacted eight-cell embryos, indicating that proviral insertion resulted in a recessive lethal defect whose phenotype was manifest very early in development. Molecular cloning of the Mpv 20 insertion site revealed that the provirus had disrupted the Npat gene, a gene of unknown function, resulting in the production of a truncated Npat mRNA. Expression of the closely linked Atm gene was found to be unaffected by the provirus.

Expression of the recessive glomerulosclerosis gene Mpv17 regulates MMP-2 expression in fibroblasts, the kidney, and the inner ear of mice.

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

Gain of Bcl-2 is more potent than bax loss in regulating mammary epithelial cell survival in vivo.

The impact of gain of Bcl-2 function on mammary epithelial cell survival was compared with loss of Bax function during the two stages of mammary gland involution. Bcl-2 gain of function reduced apoptosis 50% during the first stage and increased cell survival 70% during the second stage. Complete loss of Bax reduced apoptosis by 20% during the first stage without second stage effect. Partial loss of Bax was ineffective but increased cell survival 2.4-fold when combined with Bcl-2 gain. Gain of Bcl-2 function is more potent than loss of Bax function in regulating mammary epithelial cell survival in vivo.

Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc-induced tumorigenesis of the mammary gland in transgenic mice.

Expression of the apoptosis-inhibitory protein Bcl-2 has frequently been detected in human cancer including mammary carcinoma. The functional significance of its expression has been well established in experimental tumors of the lymphoid system, however, remains to be elucidated for epithelial tumors. In order to assess the role of Bcl-2 in mammary tumorigenesis we have generated WAP-bcl-2 transgenic mice. The strong overexpression of Bcl-2 in lactating mammary glands was preserved during early postlactational involution and apoptosis of alveolar epithelial cells was prevented without influencing the dedifferentiation of the milk-producing epithelium. Although Bcl-2 overexpression was not sufficient to induce spontaneous tumors it, however, led to an accelerated development of MMTV myc transgene-induced mammary tumors. In the mammary glands of MMTV myc transgenic mice, a high proportion of apoptotic cells was detected which was significantly reduced in the mammary glands of WAP-bcl-2/ MMTV myc double transgenic mice. Taken together, these results suggest that Bcl-2 contributes to mammary tumorigenesis by inhibiting apoptosis.

Loss of anti-mitotic effects of Bcl-2 with retention of anti-apoptotic activity during tumor progression in a mouse model.

Bcl-2 is an anti-apoptotic and anti-proliferative protein over-expressed in several different human cancers including breast. Gain of Bcl-2 function in mammary epithelial cells was superimposed on the WAP-TAg transgenic mouse model of breast cancer progression to determine its effect on epithelial cell survival and proliferation at three key stages in oncogenesis: the initial proliferative process, hyperplasia, and cancer. During the initial proliferative process, Bcl-2 strongly inhibited both apoptosis and mitotic activity. However as tumorigenesis progressed to hyperplasia and adenocarcinoma, the inhibitory effects on mitotic activity were lost. In contrast, anti-apoptotic activity persisted in both hyperplasias and adenocarcinomas. These results demonstrate that the inhibitory effect of Bcl-2 on epithelial cell proliferation and apoptosis can separate during cancer progression. In this model, retention of anti-apoptotic activity with loss of anti-proliferative action resulted in earlier tumor presentation.

A transcription enhancer acts in vitro over distances of hundreds of base-pairs on both circular and linear templates but not on chromatin-reconstituted DNA.

We have analyzed the effect of nucleosome formation and of the simian virus (SV40) enhancer on the efficiency of in vitro transcription. In a whole cell extract made from HeLa cells, nucleosome assembly on DNA results in the formation of chromatin-like complexes. However, transcription was detectable only when the DNA templates were partially or totally depleted of nucleosomes. On nucleosome-free templates, when the SV40 enhancer was present upstream from the complete SV40 early or rabbit beta-globin promoters, there was a five- to tenfold stimulation of specific transcription. When present upstream from its homologous promoter, the SV40 enhancer activated SV40 early transcription independently of its orientation with respect to the coding sequence. Point mutations known to impair the SV40 enhancer function in vivo had a similar effect in vitro. The extent of the enhancing effect was the same with linear or circular templates. When the SV40 enhancer was inserted upstream from the rabbit beta-globin gene, the activation of transcription was reduced with increasing distance between the enhancer and beta-globin upstream promoter elements, but was still significant over a distance of more than 400 base-pairs.

The human papillomavirus type 11 upstream regulatory region triggers hair-follicle-specific gene expression in transgenic mice.

We have generated transgenic mice carrying the URR of the human papillomavirus type 11 ligated in front of the Escherichia coli beta-galactosidase coding region sequence. Using X-Gal staining to demonstrate beta-galactosidase production, we observed a hair-specific transcription of the reporter gene. This transcription was limited to the epithelial cells of the hair bulge region. The transgene was developmentally regulated, as no LacZ staining was demonstrated during embryogenesis and specific staining was first observed after birth. Surprisingly, dexamethasone and ultraviolet B, but not phorbol myristate acetate or progesterone treatment of the animals resulted in an increase in number and intensity of hair follicles expressing the reporter gene.

Age-dependent hypertension in Mpv17-deficient mice, a transgenic model of glomerulosclerosis and inner ear disease.

The mutant mouse strain Mpv17-/-, carries a retroviral germline integration that inactivates the Mpv17 gene. Mpv17-deficient mice develop progressive glomerulosclerosis and sensineural deafness at early age. Characteristic basement membrane alterations are found in both sites of pathology. Mpv17 is a peroxisomal protein involved in the metabolism of reactive oxygen species, yet its molecular function is unknown. Dysregulation of antioxidant enzymes and basal membrane components has been established in this model and successful therapeutic intervention with antioxidants prove the causal role of reactive oxygen species in the development of the disease phenotype. We here investigated if the Mpv17-/- mice might be hypertensive. Indeed, our study revealed that Mpv17-/- mice developed significant systemic hypertension and tachycardia between 4 weeks and 5 months of age, accompanied by polyuria and elevated natriuresis. Judging from serum and urine parameters, the hypertensive condition develops concomitantly with the renal disease. Biochemical and pharmacological studies that used the endothelin receptor antagonist bosentan and the angiotensin converting enzyme inhibitor cilazapril indicated no involvement of the endothelin and renin-angiotensin systems in this hypertension, suggesting a potential novel mechanism of blood pressure regulation in this new murine hypertension model. Thus, Mpv17-/- mice unravel an intriguing new association between a defect in reactive oxygen metabolism and the age-dependent development of hypertension.

Ultrastructural and physiological defects in the cochlea of the Mpv17 mouse strain. A comparison between young and old adult animals.

Ultrastructural investigations were performed in young (approximately 2 months) and old (7 months) Mpv17-negative and wild-type mice. The onset, the severity and the pattern of the degeneration significantly differed between both mice strains. In the wild-type mouse strain the degenerative changes of the cochlear structures were similar to the aging pattern described for other species. In contrast, the Mpv17 mutants showed degenerative changes of the cochlear structures already at the age of 2 months. The degenerative changes were patchy arranged throughout the entire length of the cochlea and involved the organ of Corti as well as the stria vascularis epithelia with alterations of the basement membrane of the capillaries. The severe sensorineural hearing loss and degenerative changes of the cochlear structures indicate that cochlear structures, especially the outer hair cells and the intermediate cells of the stria vascularis, are vulnerable to the missing Mpv17 gene product.

Loss of auditory function in transgenic Mpv17-deficient mice.

The transgenic mouse strain Mpv17 develops severe morphological degeneration of the inner ear and nephrotic syndrome at a young age (Meyer zum Gottesberge et al., 1996; Weiher et al., 1990). The audiograms (1-32 kHz) of Mpv17-negative mice were determined from auditory brain stem responses in young (2 months) and old (7 months) animals. Audiograms of age-matched wild-type mice with the same genetic background, but wild-type at the Mpv17 locus, were also determined. Furthermore, young Mpv17-negative mice that carried a human Mpv17 homologue gene were studied. NMRI mice served as a reference for normal hearing. Mpv17-negative mice suffer from severe sensorineural hearing loss as early as 2 months after birth. In the old Mpv17-negative mice no responses could be elicited at all. The 2 month old wild-type mice had normal audiograms, at 7 months only high threshold responses were seen. The poor audiograms of the Mpv17-negative mice are assumed to be the functional correlate of the morphological degeneration of the cochlea described earlier (Meyer zum Gottesberge et al., 1996). The finding that 2 out of 4 Mpv17-negative mice with the human Mpv17 gene had normal audiograms, shows that the gene inactivation can be functionally compensated by the human Mpv17 gene product.

Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.

A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands of a DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C leads to T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.

An enhancer sequence from bovine papilloma virus DNA consists of two essential regions.

A comprehensive structural analysis of an enhancer sequence from bovine papilloma virus DNA is presented based on the construction and functional analysis of 20 mutant derivatives. The results, obtained in CV-1 tissue culture cells, show that this enhancer is a small genetic element--only 40 bp in length--that contains two essential regions. The two regions exhibit homology to each other and to DNA fragments from other viral genomes that also act as enhancers in the assay used. However, there is a certain latitude in the sequences that have enhancer activity in CV-1 cells, even within the critical regions. The results are discussed with respect to the model that enhancers are binding sites for tissue specific transcription factors. The formation of Z-DNA might be involved in the enhancement process. However, single base pair transitions in an 8 base pair stretch of alternating purines and pyrimidines within the BPV enhancer which conserve this pattern destroy enhancer function.

Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences.

A bacterial suppressor tRNA gene was introduced into the long terminal repeat of the Moloney murine leukemia virus (Mo-MuLV) proviral genome to construct a retrovirus that allows easy cloning of the provirus with flanking host sequences. A replication competent virus, Mo-MuLV sup containing a tRNA amber suppressor gene, was derived that replicates to high titers in tissue culture cells and stably transduces the bacterial gene. The recombinant virus can efficiently replicate in vivo when microinjected into midgestation embryos or when injected into newborn mice and displays the same tissue tropism as wild-type Mo-MuLV. The suppressor gene in Mo-MuLV sup is functional in bacteria and allows efficient recovery of proviral genomes. This was shown by ligation of DNA from infected cells to phage lambda Charon 4A arms and selective growth of recombinant phages on su- host cells. All recovered phages contained Mo-MuLV proviral sequences and, because of the high cloning capacity of phage lambda, 1-11 kilobases of flanking host DNA. This virus should facilitate studying virus-host interactions in tissue culture cells and in animals.

Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells.

Moloney murine leukemia virus (M-MuLV) and M-MuLV-derived retroviral vectors are not expressed in early mouse embryos or in embryonal carcinoma cells. M-MuLV-derived mutants or M-MuLV-related variants which transduce the neomycin phosphotransferase gene can, however, induce drug resistance in embryonal carcinoma cells with high efficiency. In this study we investigated the sequences critical for retroviral gene expression in two different embryonal carcinoma cell lines, F9 and PCC4. We show that two synergistically acting sequence elements mediate expression in embryonal carcinoma cells. One of these is located within the U3 region of the viral long terminal repeat, and the second one is in the 5' untranslated region of the retrovirus. The latter element, characterized by a single point mutation, affects the level of stable RNA in infected cells, suggesting a regulatory mechanism similar to that of human immunodeficiency virus in human T cells.

Avian erythroblastosis virus E26: nucleotide sequence of the tripartite onc gene and of the LTR, and analysis of the cellular prototype of the viral ets sequence.

An intact 5.7-kb provirus of the avian erythroblastosis virus E26 has been molecularly cloned for comparisons with avian myeloblastosis virus (AMV) and other avian tumor viruses. E26 and AMV transform hemopoietic cells exclusively. Both cause myeloblastosis, but E26 also causes erythroblastosis. Sequence analysis of the proviral DNA showed that: The tripartite transforming gene of E26 forms a contiguous reading frame of 1046 codons, including 272 gag, 283 mybE, and 491 ets codons. No subgenomic ets-specific mRNA was detected in E26-infected cells. By contrast, the onc gene of AMV consists almost entirely of a mybA sequence expressed via subgenomic mRNA that extends over the 5' and 3' ends of mybE. mybE is only slightly diverged from the mybA homolog of AMV and even less from the cellular proto-myb sequence with no characteristic mutation that sets apart the two viruses from proto-myb. The U5 region of the long terminal repeat (LTR) of E26 and AMV are colinear and differ only in scattered point mutations. The U3 region of the E26 LTR is different from that of AMV but is colinear and closely related with that of avian carcinoma virus MH2 and also with that of Prague Rous sarcoma virus (RSV), except for an unexpected 16-nucleotide substitution of 22 RSV nucleotides. Upstream of the 3' LTR, the c region of E26 appears to be the same as that of RSV for 70 nucleotides and very similar to those of AMV and MH2 for about 20 to 30 nucleotides. Since the U3s of E26, MH2 and RSV are very closely related and neither MH2 nor RSV show a particular erythroblast tropism, it is possible that the U3 does not play a critical role in the erythroblast tropism of E26. Electrophoretic size analyses of chicken DNA digested with restriction enzymes indicate that DNA fragments totaling over 50 kb hybridize with viral ets DNA.

Glomerular overproduction of oxygen radicals in Mpv17 gene-inactivated mice causes podocyte foot process flattening and proteinuria: A model of steroid-resistant nephrosis sensitive to radical scavenger therapy.

Focal segmental glomerulosclerosis is a steroid-resistant glomerular disease characterized by foot process flattening and heavy proteinuria. A similar disease was found to occur spontaneously in mice in which the Mpv17 gene was inactivated by retroviral insertion (Mpv17-/- mice). Here evidence is provided that glomerular damage in this murine model is due to overproduction of oxygen radicals and accumulation of lipid peroxidation adducts that were found in isolated glomeruli of Mpv17-/- mice. The development of glomerular disease in Mpv17-/- mice was inhibited by scavengers of oxygen radicals (dithiomethylurea) and lipid peroxidation (probucol), but not by steroid treatment. Although the glomerular polyanion was greatly reduced in proteinuric Mpv17-/- mice, it was preserved by antioxidative therapy. These results indicate that the glomerular disease in Mpv17-/- mice qualifies as a model of steroid-resistant focal segmental glomerulosclerosis and that experimental therapies with scavengers of oxygen radicals and lipid peroxidation efficiently ameliorate glomerular damage.

Inner ear defect similar to Alport's syndrome in the glomerulosclerosis mouse model Mpv17.

The Mpv17 mouse strain is a recessive transgenic mouse mutant that develops glomerulosclerosis and nephrotic syndrome at a young age. The phenotype results from a loss of function of a gene coding for a hydrophobic peroxisomal protein of 176 amino acids of 20 kDa following its destruction by retroviral integration. To investigate a potential effect of the missing Mpv17 function on the inner ear light and electron microscopic investigations were performed on the inner ears of Mpv17 mice and controls. These revealed degeneration of the stria vascularis and spiral ligament, loss of cochlear neurons and degeneration of the organ of Corti. The alterations observed here were similar to those described for Alport's syndrome, an inherited disorder characterized by progressive nephritis and neurosensory deafness. These findings indicate that although the molecular cause is different, the Mpv17 mouse model may share pathological mechanisms involved in patients with Alport's syndrome. At present the Mpv17 mouse appears to be a suitable animal model for this disease and may help to further elucidate the relationship between the kidney and the inner ear.