Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.
(49/7062)
Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer/promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15-20 microgram/ml of canine factor IX was detected in the plasma of mice injected with 5.6 x 10(11) particles of an AAV vector for >5 months. The activated partial thromboplastin time of the treated mice was fully corrected to higher than normal levels. Liver-specific expression of canine factor IX was confirmed by immunofluorescence staining, and secreted factor IX protein was identified in the mouse plasma by Western blotting. All treated mice survived the tail clip test without difficulty. Thus, a single intraportal injection of a recombinant adeno-associated virus vector expressing factor IX successfully cured the bleeding disorder of hemophilia B mice, proving the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases. (+info)
Ectopic expression of prion protein (PrP) in T lymphocytes or hepatocytes of PrP knockout mice is insufficient to sustain prion replication.
(50/7062)
The cellular form of the Prion protein (PrPC) is necessary for prion replication in mice. To determine whether it is also sufficient, we expressed PrP under the control of various cell- or tissue-specific regulatory elements in PrP knockout mice. The interferon regulatory factor-1 promoter/Emu enhancer led to high PrP levels in the spleen and low PrP levels in the brain. Following i.p. scrapie inoculation, high prion titers were found in the spleen but not in the brain at 2 weeks and 6 months, showing that the lymphoreticular system by itself is competent to replicate prions. PrP expression directed by the Lck promoter resulted in high PrP levels on T lymphocytes only but, surprisingly, did not allow prion replication in the thymus, spleen, or brain following i.p. inoculation. A third transgenic line, which expressed PrP in the liver under the control of the albumin promoter/enhancer-albeit at low levels-also failed to replicate prions. These results show that expression of PrP alone is not sufficient to sustain prion replication and suggest that additional components are needed. (+info)
Fine-scale transgenic mapping of the MyoD core enhancer: MyoD is regulated by distinct but overlapping mechanisms in myotomal and non-myotomal muscle lineages.
(51/7062)
Skeletal muscle lineage determination is regulated by the myogenic regulatory genes, MyoD and Myf-5. Previously, we identified a 258 bp core enhancer element 20 kb 5' of the MyoD gene that regulates MyoD gene activation in mouse embryos. To elucidate the cis control mechanisms that regulate MyoD transcription, we have mutagenized the entire core enhancer using linker-scanner mutagenesis, and have tested the transcriptional activity of enhancer mutants using lacZ reporter gene expression in transgenic mouse embryos. In total, 83 stable transgenic lines representing 17 linker-scanner mutations were analyzed in midgestational mouse embryos. Eight linker-scanner mutations resulted in a partial or complete loss of enhancer activity, demonstrating that MyoD is primarily under positive transcriptional control. Six of these mutations reduced or abolished transgene expression in all skeletal muscle lineages, indicating that activation of MyoD expression in trunk, limb and head musculature is regulated, in part, by shared transcriptional mechanisms. Interestingly, however, two adjacent linker-scanner mutations (LS-14 and LS-15) resulted in a dramatic reduction in transgene expression specifically in myotomes at 11.5 days. At later stages, transgene expression was absent or greatly reduced in myotomally derived muscles including epaxial muscles (deep back muscles) and hypaxial muscles of the body wall (intercostal muscles, abdominal wall musculature). In contrast, head muscles, as well as muscles of the body derived from migrating muscle progenitor cells (e.g. limb, diaphragm), were unaffected by these mutations. In Pax-3-mutant mice, LS-14 and LS-15 transgene expression was eliminated in the body, but was unaffected in the head, yielding an identical expression pattern to the endogenous MyoD gene in mice mutant for both Myf-5 and Pax-3. These data support the hypothesis that LS-14 and LS-15 define the core enhancer targets for Myf-5-dependent activation of MyoD in myotomal muscles. (+info)
Trans-acting factors required for inclusion of regulated exons in the Ultrabithorax mRNAs of Drosophila melanogaster.
(52/7062)
Alternatively spliced Ultrabithorax mRNAs differ by the presence of internal exons mI and mII. Two approaches were used to identify trans-acting factors required for inclusion of these cassette exons. First, mutations in a set of genes implicated in the control of other alternative splicing decisions were tested for dominant effects on the Ubx alternative splicing pattern. To identify additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested first for dominant enhancement of the haploinsufficient Ubx haltere phenotype and second for effects on the splicing pattern. Inclusion of the cassette exons in Ubx mRNAs was reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in control of P-element splicing. Significant reductions of mI and mII inclusion were also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and crooked neck. The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural similarities to yeast-splicing factors Prp39p and Prp42p. Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII. Possible roles of identified factors are discussed in the context of the resplicing strategy for generation of alternative Ubx mRNAs. (+info)
Recognition of multiple patterns of DNA sites by Drosophila homeodomain protein Bicoid.
(53/7062)
Our previous studies demonstrated that the Drosophila homeodomain protein, Bicoid (Bcd), binds DNA cooperatively. In this study, we determined the patterns of adjacent DNA sites required for cooperative recognition by Bcd. Our in vitro selection and biochemical experiments demonstrated that Bcd binds preferentially to both head-to-head and tail-to-tail symmetric sites that are separated by short spacing. An increase in the spacing reduces the strict requirement of symmetric patterns of adjacent sites, permitting Bcd to recognize tandem repeat sites cooperatively. Our further experiments in vivo showed that the only pair of optimally spaced symmetric Bcd sites in a hunchback (hb) enhancer element contributes the most to transcriptional activation by Bcd, demonstrating the biological importance of the binding site patterns revealed by our in vitro selection studies. (+info)
The promoter and the enhancer region of the KLK 3 (prostate specific antigen) gene is frequently mutated in breast tumours and in breast carcinoma cell lines.
(54/7062)
KLK3 or prostate specific antigen (PSA) is a serine protease, which is an established tumour marker of prostatic adenocarcinoma. PSA is now used widely for the diagnosis and monitoring of patients with prostate cancer. Recent studies have demonstrated that about 70% of breast cancers produce PSA. In this study, we examined the molecular mechanism underlying the expression of the PSA gene in breast cancer and breast cancer cell lines. We analysed nine breast tumours categorized on the basis of high- or low-PSA expression in tumour cytosols and four breast cancer cell lines. To determine abnormalities associated with PSA expression in breast tumours, genomic DNA was extracted and all five exons of the PSA gene were polymerase chain reaction (PCR) amplified and sequenced on both strands. PCR amplification was also performed for the promoter and enhancer elements of the PSA gene. No mutations were observed in the coding portion of the gene. A polymorphism was observed in exon 2 from three breast tumours. However, sequencing of the promoter and the enhancer elements of the PSA gene reveals several point mutations. Within a 5.8-kb promoter/enhancer region of the PSA gene, we detected 16 different mutational hotspots (appearing more than once in the nine tumours). Among these hotspots, two appeared in seven out of nine tumours. Most importantly, the androgen response element (ARE I) in the proximal promoter was found mutated in four tumours and in the breast carcinoma cell line MCF-7. Mutations associated with the ARE I have been shown previously to result in an 80% decrease in PSA gene expression. The mutations in the core enhancer and promoter region probably contribute to the aberrant expression of the PSA gene in breast tumours, possibly by altering the regulation of the gene by steroid hormones. (+info)
SPI-B activates transcription via a unique proline, serine, and threonine domain and exhibits DNA binding affinity differences from PU.1.
(55/7062)
SPI-B is a B lymphocyte-specific Ets transcription factor that shares a high degree of similarity with PU.1/SPI-1. In direct contrast to PU.1(-/-) mice that die in utero and lack monocytes, neutrophils, B cells, and T cells, Spi-B-/- mice are viable and exhibit a severe B cell proliferation defect. Since PU.1 is expressed at wild type levels in Spi-B-/- B cells, the mutant mice provide genetic evidence that SPI-B and PU.1 have at least some non-redundant roles in B lymphocytes. To begin to understand the molecular basis for these defects, we delineated functional domains of SPI-B for comparison to those of PU.1. By using a heterologous co-transfection system, we identified two independent transactivation domains in the N terminus of SPI-B. Interestingly, only one of these domains (amino acids 31-61), a proline/serine/threonine-rich region, unique among Ets proteins, is necessary for transactivation of the immunoglobulin lambda light chain enhancer. This transactivation motif is in marked contrast to PU.1, which contains acidic and glutamine-rich domains. In addition, we describe a functional PU.1 site within the c-FES promoter which SPI-B fails to bind efficiently and transactivate. Finally, we show that SPI-B interacts with the PU.1 cofactors Pip, TBP, c-Jun and with lower affinity to nuclear factor interleukin-6beta and retinoblastoma. Taken together, these data suggest that SPI-B binds DNA with a different affinity for certain sites than PU.1 and harbors different transactivation domains. We conclude that SPI-B may activate unique target genes in B lymphocytes and interact with unique, although currently unidentified, cofactors. (+info)
Identification of the transcriptional regulatory sequences of human kallikrein 2 and their use in the construction of calydon virus 764, an attenuated replication competent adenovirus for prostate cancer therapy.
(56/7062)
Human glandular kallikrein (hK2) and prostate-specific antigen (PSA) are related members of the human kallikrein gene family. The genes for hK2 and PSA are expressed predominately in the prostate, are transcriptionally up-regulated by androgens, and share 78% homology. Previously, one functional androgen response element was identified within the proximal promoter (-324 to +33 relative to the cap site) of the hK2 gene. To detect additional upstream regulatory elements, the 12.3 kbp between the PSA gene and 5' to the hK2 gene were amplified by PCR and linked to a promoterless firefly luciferase reporter gene. Transient transfection experiments showed an androgen-dependent enhancer, located between -3.4 and -5.2 kb upstream of the transcription start site of the hK2 gene. This hK2 enhancer increased luciferase expression 100-fold in the presence of the testosterone analogue R1881. The hK2 enhancer contains an androgen response element that lost activity when mutated. The hK2 enhancer/promoter demonstrated activity in PSA(+) LNCaP cells whereas the enhancer/promoter was inactive in PSA(-) 293, A549, HBL100, HUH-7, LoVo, MCF-7, OVCAR-3, and PC-3 cells. Insertion of the hK2 enhancer/promoter into adenovirus to drive the E1A genes of adenovirus type 5 (Ad5) created an attenuated replication competent adenovirus variant Calydon virus (CV) 763, which replicates similarly to wild-type adenovirus in prostate tumor cells but is attenuated in nonprostate tumor cells. In addition, CV764, an adenovirus variant containing the previously cloned prostate-specific enhancer (to drive the Ad5 E1A genes) and the hK2 enhancer/promoter (to drive the Ad5 E1B genes) was constructed. CV764 is significantly attenuated and has a high therapeutic index with a cell specificity of 10,000:1 for PSA(+) LNCaP cells, compared to ovarian cancer OVCAR-3 cells and SK-OV-3 cells and PA-1 cells. CV764 is also highly attenuated in primary human microvascular endothelial cells. (+info)