The murine cataractogenic mutation, Cat Fraser, segregates independently of the gamma crystallin genes

The murine mutation, Cat Fraser (Cat(Fr)), causes dominantly inherited ocular cataracts. Lenses of adult mice bearing this mutation contain reduced amounts of all seven γ-crystallin proteins and their corresponding transcripts. Levels of other lens proteins and transcripts appear normal and no extra-ocular effects of the mutation have been observed. The selective effect of this mutation on the γ-crystallins is consistent with the possibility that the site at which it occurs is involved in the coordinated regulation of the family of genes which encodes them. We have shown that several restriction fragment length polymorphisms in the γ-crystallin genes segregate independently of the Cat(Fr) mutation. Therefore, despite its selective effect on the expression of the γ-crystallin genes, the mutation is not linked to them. This observation rules out the possibility that the mutation is in a cis-acting regulatory site.published_or_final_versio

Localization of a beta-crystallin gene, Hu beta A3/A1 (gene symbol:CRYB1), to the long arm of human chromosome 17

We have assigned a human β-crystallin gene, HuβA3/A1 (gene symbol: CRYB1), to chromosome 17 using a panel of 19 human-hamster somatic cell hybrids and blot-hybridization analysis of cell hybrid DNA. Positive probe-hybridization signal was detected in a hybrid that had last the short arm of human chromosome 17 but retained the long arm, translocated to a hamster chromosome. In addition, in situ hybridization analysis of metaphase chromosome spreads of this cell line suggested that the most probable location for CRYB1 is on the long arm of chromosome 17, in the region q21.published_or_final_versio

A new mouse model for renal lesions produced by intravenous injection of diphtheria toxin A-chain expression plasmid

BACKGROUND: Various animal models of renal failure have been produced and used to investigate mechanisms underlying renal disease and develop therapeutic drugs. Most methods available to produce such models appear to involve subtotal nephrectomy or intravenous administration of antibodies raised against basement membrane of glomeruli. In this study, we developed a novel method to produce mouse models of renal failure by intravenous injection of a plasmid carrying a toxic gene such as diphtheria toxin A-chain (DT-A) gene. DT-A is known to kill cells by inhibiting protein synthesis. METHODS: An expression plasmid carrying the cytomegalovirus enhancer/chicken β-actin promoter linked to a DT-A gene was mixed with lipid (FuGENE™6) and the resulting complexes were intravenously injected into adult male B6C3F1 mice every day for up to 6 days. After final injection, the kidneys of these mice were sampled on day 4 and weeks 3 and 5. RESULTS: H-E staining of the kidney specimens sampled on day 4 revealed remarkable alterations in glomerular compartments, as exemplified by mesangial cell proliferation and formation of extensive deposits in glomerular basement membrane. At weeks 3 and 5, gradual recovery of these tissues was observed. These mice exhibited proteinuria and disease resembling sub-acute glomerulonephritis. CONCLUSIONS: Repeated intravenous injections of DT-A expression plasmid DNA/lipid complex caused temporary abnormalities mainly in glomeruli of mouse kidney. The disease in these mice resembles sub-acute glomerulonephritis. These DT-A gene-incorporated mice will be useful as animal models in the fields of nephrology and regenerative medicine

Treatment of ovarian cancer ascites by intra-peritoneal injection of diphtheria toxin A chain-H19 vector: a case report

<p>Abstract</p> <p>Introduction</p> <p>Ovarian cancer ascitic fluid, which contains malignant cells, is usually present in women with an advanced stage disease. There are currently no effective therapies for the treatment of ovarian cancer ascitic fluid. We developed a new therapeutic strategy to target expression of the diphtheria toxin fragment A gene in ovarian tumor cells under the control of H19 regulatory sequences.</p> <p>Case presentation</p> <p>A 64-year-old Caucasian woman was diagnosed with a stage IIIc epithelial ovarian cancer. She suffered from progressive disease, accumulation of malignant ascites that needed to be drained weekly, abdominal pain, vomiting, anorexia and severe weakness. Infusion of the diphtheria toxin A chain-H19 plasmid into the peritoneum of our patient resulted in complete resolution of the ascites with minimum adverse events.</p> <p>Conclusion</p> <p>On the basis of this preliminary experience, we are currently conducting an extensive Phase I study on a larger number of patients in order to assess the safety and preliminary efficacy of this novel patient-oriented treatment approach.</p

Development of targeted therapy for ovarian cancer mediated by a plasmid expressing diphtheria toxin under the control of H19 regulatory sequences

<p>Abstract</p> <p>Background</p> <p>Ovarian cancer ascites fluid (OCAF), contains malignant cells, is usually present in women with an advanced stage disease and currently has no effective therapy. Hence, we developed a new therapy strategy to target the expression of diphtheria toxin gene under the control of H19 regulatory sequences in ovarian tumor cells. H19 RNA is present at high levels in human cancer tissues (including ovarian cancer), while existing at a nearly undetectable level in the surrounding normal tissue.</p> <p>Methods</p> <p>H19 gene expression was tested in cells from OCAF by the in-situ hybridization technique (ISH) using an H19 RNA probe. The therapeutic potential of the toxin vector DTA-H19 was tested in ovarian carcinoma cell lines and in a heterotopic animal model for ovarian cancer.</p> <p>Results</p> <p>H19 RNA was detected in 90% of patients with OCAF as determined by ISH. Intratumoral injection of DTA-H19 into ectopically developed tumors caused 40% inhibition of tumor growth.</p> <p>Conclusion</p> <p>These observations may be the first step towards a major breakthrough in the treatment of human OCAF, while the effect in solid tumors required further investigation. It should enable us to identify likely non-responders in advance, and to treat patients who are resistant to all known therapies, thereby avoiding treatment failure.</p

Replication of pSV2-gpt in COS-1 cells: Stability of plasmid DNA in the presence and absence of biochemical selection

We have previously demonstrated that COS-1 cell lines transformed by pSV2-gpt and maintained under biochemical selection replicate multiple copies of extrachromosomal plasmid DNA (1). We have now examined the replication and stability of this DNA in a representative cell line. In situ hybridization analyses revealed that intense replication of pSV2-gpt occurs in only a small subpopulation of cells and results from bursts of plasmid replication that occur periodically and spontaneously in the cell population. This suggests that COS-1 cells are only semipermissive for pSV2-gpt replication. No correlation was observed between levels of pSV2-gpt replication and the presence or absence of biochemical selection for the Gpt marker. However, growth of cells under nonselective conditions led to a rapid and progressive loss of pSV2-gpt DNA. This loss correlated with segregation of Gpt- revertants that lacked detectable plasmid sequences. Hence, maintenance of pSV2-gpt in the cell line was dependent on continuous biochemical selection. Stable replication of pSV2-gpt could be observed as late as four months after transfection, suggesting that this system might be useful for propagation of cloned DNA in COS-1 cells for extended periods of time. However, by nine months, extensive rearrangements of pSV2-gpt sequences were detected, indicating ultimate instability of the plasmid in the host cells.link_to_subscribed_fulltex

A frameshift mutation in the γE-crystallin gene of the Elo mouse

The murine Elo (eye lens obsolesence) mutation confers a dominant phenotype characterized by malformation of the eye lens. The mutation maps to chromosome 1, in close proximity to the γE-crystallin gene which is the 3′-most member of the γ-crystallin gene cluster. We have analysed the sequence of this gene from the Elo mouse and identified a single nucleotide deletion which destroys the fourth and last "Greek key" motif of the protein. This mutation is tightly associated with the phenotype, as no recombination was detected in 274 meioses. In addition, the mutant mRNA is present in the affected lens, providing further support for our hypothesis that the deletion is responsible for the dominant Elo phenotype.link_to_subscribed_fulltex

γ-Crystallin family of the mouse lens: Structural and evolutionary relationships

The heterogeneity inherent among γ-crystallins of the mouse lens was investigated by sequence analysis of three γ-crystallin-specific cDNAs. Comparison of the nucleotide sequence of these cDNAs and one previously reported by us revealed that the four γ-cDNAs share 80-90% homology in nucleotide sequence. The entire 3' half of the coding region shows more variability than the 5's half, whereas the greatest variability is observed in the 3' untranslated region where numerous base substitutions, deletions, and insertions seem to have occurred. Alignment of the amino acid sequences of the four mouse γ-crystallins according to the known four structural motifs of the major calf γ-crystallin, γ-II, suggests that all four mouse polypeptides are structurally very similar to calf γ-II. However, most of the mouse polypeptides differ from γ-II by the absence of one amino acid residue, resulting in a shorter connecting peptide between the two globular domains of the protein. Primary sequence alignment also revealed that the four mouse γ-crystallins are most divergent in the third structural motif of the polypeptide. The significance of these differences in terms of the structure and function of the γ-crystallins in the mouse lens is discussed.published_or_final_versio

Structural and evolutionary relationships among five members of the human γ-crystallin gene family

We have characterized five human γ-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.published_or_final_versio

Temporal regulation of six crystallin transcripts during mouse lens development

Using the polymerase chain reaction (PCR) and RNA blot analysis. we have examined the differential expression patterns of the γ-crystallins during lens development. Since only four of these genes had been previously characterized, the cDNAs for the remaining two genes. γC and γF, were isolated and sequenced. The steady-state mRNA profiles were then determined by RNA blot analysis of samples from embryonic stages to 180 days after birth, with gene-specific probes for γA, γB, γC, and γD, and a common probe for γE and γF. Due to the paucity of mismatches between the γE and γF-crystallin genes, the PCR technique was exploited to determine their relative abundance. The data showed that while all six γ-crystallin genes were expressed in the embryonic lens, they were differentially regulated during development. At early stages, the levels of γB and γC mRNAs were found to be relatively low in comparison to those for γA, γD, γE and γF. After 30-40 days however, the levels of γA, γE, and γF mRNAs declined rapidly, and the γB, γC and γD transcripts became the major γ-crystallin mRNA species. The utility of the PCR technique in studying the relative abundance of steady-state γ-crystallin mRNAs was also investigated.link_to_subscribed_fulltex