Large genomic fibrillin-1 (FBN1) gene deletions provide evidence for true haploinsufficiency in Marfan syndrome

Mutations in the FBN1 gene are the major cause of Marfan syndrome (MFS), an autosomal dominant connective tissue disorder, which displays variable manifestations in the cardiovascular, ocular, and skeletal systems. Current molecular genetic testing of FBN1 may miss mutations in the promoter region or in other noncoding sequences as well as partial or complete gene deletions and duplications. In this study, we tested for copy number variations by successively applying multiplex ligation-dependent probe amplification (MLPA) and the Affymetrix Human Mapping 500 K Array Set, which contains probes for approximately 500,000 single-nucleotide polymorphisms (SNPs) across the genome. By analyzing genomic DNA of 101 unrelated individuals with MFS or related phenotypes in whom standard genetic testing detected no mutation, we identified FBN1 deletions in two patients with MFS. Our high-resolution approach narrowed down the deletion breakpoints. Subsequent sequencing of the junctional fragments revealed the deletion sizes of 26,887 and 302,580 bp, respectively. Surprisingly, both deletions affect the putative regulatory and promoter region of the FBN1 gene, strongly indicating that they abolish transcription of the deleted allele. This expectation of complete loss of function of one allele, i.e. true haploinsufficiency, was confirmed by transcript analyses. Our findings not only emphasize the importance of screening for large genomic rearrangements in comprehensive genetic testing of FBN1 but, importantly, also extend the molecular etiology of MFS by providing hitherto unreported evidence that true haploinsufficiency is sufficient to cause MFS.

Cloning and characterization of the genes involved in cambial growth in response to elevated [CO2]

Aspen (Populus tremuloides) trees growing under elevated [CO2] at a free-air CO2 enrichment (FACE) site have produced significantly more biomass compared to control trees. The molecular mechanisms underlying the observed increase in biomass productivity was investigated by producing transcriptomic profiles of the vascular cambium zone (VCZ) and leaves, followed by a comparative study to identify genes and pathways that showed significant changes following long-term exposure to elevated [CO2]. This study is mainly to verify if genetic modification of a few selected candidate genes including CAP1, CKX6, and ASML2 that are expressed in vascular cambium in response to elevated [CO2] can cause the changes in plant growth and development. To this end, these three genes were cloned into both sense and antisense constructs. Then antisense and sense transgenic lines of above-mentioned genes were developed. 15 events were generated for 5 constructs, which were confirmed with regular PCR and RT-PCR. Confirmed plants were planted in greenhouse for growth and phenotypic characterization. The expression of CAP1, CKX6 and ASML2 in antisense plants was measured by real-time RT-PCR, and the changes caused by gene interference in cambial growth were studies by analyzing the microscopic sections made from the antisense transgenic plants. It has been found that 1) CAP1 is mainly expressed in xylem and root. 2) RNAi suppression of CAP1 significantly affected height and diameter. 3) CAP1, ASML2 and CKX6 affected xylem and phloem cell proliferation and elongation. Due to the delay in regenerating sense transgenic plants, the characterization of sense transgenic plants is limited to growth only.

Cloning and characterization of a novel zinc finger protein (rZFP96) in the rat corpus luteum

The corpus luteum (CL) is a temporary organ involved in the maintenance of pregnancy. In the course of its life-cycle, the CL undergoes two distinct and consecutive processes for its inevitable removal through apoptosis: functional and structural luteolysis. We isolated a gene encoding for a novel rat zinc finger protein (ZFP), named rat ZFP96 (rZFP96) from an ovarian lambda cDNA library. Sequence analysis revealed close sequence and structural similarity to mouse ZFP96 and human zinc finger protein 305 (ZNF305). Quantitative reverse transcription-polymerase chain reaction analysis revealed a positive correlation with the end of pregnancy, that is, the onset of structural luteolysis of the CL. Messenger RNA levels increased 3-fold (P < 0.01) between days 13 and 22 of pregnancy and 8-fold (P < 0.01) between day 13 of pregnancy and day 1 post-partum. In addition, we detected rZFP96 expression in mammary, placenta, heart, kidney and skeletal muscle. Sequence analysis predicted that rZFP96 has a high probability of localizing to the nuclear compartment. The presence of both a perfect consensus TGEKP linker sequence between zinc fingers 2 and 3 as well as several similar sequences between the other zinc fingers suggests physical interaction with DNA. Speculatively, rZFP96 may therefore function as a transcription factor, switching-off pro-survival genes and/or upregulating pro-apoptotic genes and thereby contributing to the demise of the CL.

Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae

Phylogenies of housekeeping gene and 16S rRNA gene sequences were compared to improve the classification of the bacterial family Pasteurellaceae and knowledge of the evolutionary relationships of its members. Deduced partial protein sequences of the housekeeping genes atpD, infB and rpoB were compared in 28, 36 and 28 representative taxa of the Pasteurellaceae, respectively. The monophyly of representatives of the genus Gallibacterium was recognized by analysis of all housekeeping genes, while members of Mannheimia, Actinobacillus sensu stricto and the core group of Pasteurella sensu stricto formed monophyletic groups with two out of three housekeeping genes. Representatives of Mannheimia, Actinobacillus sensu stricto, [Haemophilus] ducreyi and [Pasteurella] trehalosi formed a monophyletic unit by analysis of all three housekeeping genes, which was in contrast to the 16S rRNA gene-derived phylogeny, where these taxa occurred at separate positions in the phylogenetic tree. Representatives of the Rodent, Avian and Aphrophilus-Haemophilus 16S rRNA gene groups were weakly supported by phylogenetic analysis of housekeeping genes. Phylogenies derived by comparison of the housekeeping genes diverged significantly from the 16S rRNA gene-derived phylogeny as evaluated by the likelihood ratio test. A low degree of congruence was also observed between the individual housekeeping gene-derived phylogenies. Estimates on speciation derived from 16S rRNA and housekeeping gene sequence comparisons resulted in quite different evolutionary scenarios for members of the Pasteurellaceae. The phylogeny based on the housekeeping genes supported observed host associations between Mannheimia, Actinobacillus sensu stricto and [Pasteurella] trehalosi and animals with paired hooves.

Cloning and characterization of two bistructural S-layer-RTX proteins from Campylobacter rectus

Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots.

Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis.

BACKGROUND: Plasmids containing hylEfm (pHylEfm) were previously shown to increase gastrointestinal colonization and lethality of Enterococcus faecium in experimental peritonitis. The hylEfm gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated E. faecium, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the hylEfm-region and we evaluated their effect on virulence using a murine peritonitis model. RESULTS: Five mutants of the hylEfm-region of pHylEfmTX16 from the sequenced endocarditis strain (TX16 [DO]) were obtained using an adaptation of the PheS* system and were evaluated in a commensal strain TX1330RF to which pHylEfmTX16 was transferred by mating; these include i) deletion of hylEfm only; ii) deletion of the gene downstream of hylEfm (down) of unknown function; iii) deletion of hylEfm plus down; iv) deletion of hylEfm-down and two adjacent genes; and v) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by cat. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHylEfmTX16Δ7,534 was transferred to the TX1330RF background, the transconjugant was affected in in vitro growth versus TX1330RF(pHylEfmTX16) and was attenuated in virulence; however, neither hylEfm nor hylEfm-down restored wild type function. We did not observe any in vivo effect on virulence of the other deletions of the hylEfm-region CONCLUSIONS: The four genes of the hylEfm region (including hylEfm) do not mediate the increased virulence conferred by pHylEfmTX16 in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of E. faecium in the future.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Isolation and analysis of regulatory and structural mutations in the {\it recA\/} gene of {\it Escherichia coli}

The recA gene is essential for homologous recombination and for inducible DNA repair in Escherichia coli. The level of recA expression is important for these functions. The growth defect of a lambda phage carrying a recA-lacZ fusion was used to select mutations that reduced recA expression. Nine of these mutations were single base changes in the recA promoter; each reduced both induced and basal (repressed) levels of expression, indicating that only one promoter is used under both circumstances. Deletion analysis of the promoter region and S1 mapping of transcripts confirmed that there is only one promoter responsible for both basal and induced expression. Some of the mutants, however, displayed a ratio of induced to repressed expression that was much lower than wild-type. For one of these mutants (recA1270) LexA binding studies showed that this was not due to a change in the affinity of LexA repressor for the operator site. The extent of binding of RNA polymerase to this mutant promoter, however, was much reduced, and the complexes formed were qualitatively different. Further binding experiments provided some evidence that LexA does not block RNA polymerase binding to the recA promoter, but inhibits a later step in initiation. Behavior of the mutants with altered induction ratios could be explained if LexA binding to the operator actually increases RNA polymerase binding to the promoter in a closed complex compensating for defects in polymerase binding caused by the mutations.^ In a study of mutations in the recA structural gene, site-directed mutagenesis was used to replace cysteine codons at positions 90, 116, and 129 with a number of different codons. In vivo analysis of the replacements showed that none of the cysteines is absolutely essential and that they do not have a direct role as catalysts in ATP hydrolysis. Some amino acid substitutions abolished all RecA functions, while a few resulted in partial or altered function. Amino acids at positions 90 and 129 tended to affect all functions equally, while the amino acid at position 116 appeared to have a particular effect on the protease activity of the protein. ^

Differential gene expression in multistep tumorigenesis using an {\it in vitro\/} human cell model

Using a human terato-carcinoma cell line, PA-1, the functional role of the oncogenes and tumor suppressor gene involved in the multistep process of carcinogenesis have been analyzed. The expression of AP-2 was strongly correlated with the susceptibility to ras transformation. The differential responsiveness to growth factors between stage 1 ras resistant cells and stage 2 ras susceptible cells was observed, indicating that the ability of stage 2 cells to respond to the mutated ras oncogenes in transformation correlated with the ability to be stimulated by certain growth factors. Using differential screening of cDNA libraries, a number of differentially expressed cDNA clones was isolated. One of those, clone 12, is overexpressed in ras transformed stage 3 cells. The amino acid sequence of clone 12 is almost identical to a mouse LLrep3 gene that was growth-regulated, and 78% similar to a yeast ribosomal protein S4. These results suggest that the S4 gene may be involved in regulation of growth. Clone 9 is expressed in stage 1 ras resistant cells (3.5-kb and 3.0-kb transcripts) but the expression of this clone in stage 2 ras susceptible cells and stage 3 ras-transformed cells is greatly diminished. The expression of this cDNA clone was increased to at least five fold in ras resistant cells and nontumorigenic hybrids treated with retinoic acid but not increased in retinoic acid treated ras susceptible cells, ras transformed cells and the tumorigenic segregants. Partial sequence of this clone showed no homology to the sequences in Genbank. These findings suggest that clone 9 could be a suppressor gene or the genes that are involved in the biochemical pathway of tumor suppression or neurogenic differentiation. The apparent pleiotropic effect of the loss of this suppressor gene function support Harris' proposal that tumor suppressor genes regulate differentiation. The tumor suppressor gene may act as negative regulator of tumor growth by controlling gene expression in differentiation. ^

Mapping and cloning of genes from portions of the human genome using somatic cell hybrids

Human x rodent somatic cell hybrids have played an important role in human genetics research. They have been especially useful for assigning genes to chromosomes and isolating DNA markers from specific regions of the human genome.^ By employing a combination of somatic cell genetic, recombinant DNA, and cytogenetic techniques, human DNA excision repair gene ERCC4 was mapped regionally to human 16p13.13-13.2, even though the gene has not been cloned. Human x Chinese hamster ovary (CHO) cell hybrids selected for human ERCC4 activity and containing 16p13.1-p13.3 as the only human genetic material were identified. These hybrids were used to order DNA markers located in 16p13.1-p13.3. New DNA markers physically close to ERCC4 were isolated from such hybrids. Using amplified human DNA from the hybrids as probe in fluorescent in situ hybridization, the short arm breakpoint in the chromosome 16 inversion associated with acute myelomonocytic leukemia (AMML) was found to be physically close to the ERCC4 gene. The physical mapping and eventually, the cloning of the ERCC4 gene, will benefit the understanding of the DNA repair system and the study of other important biomedical problems such as tumorigenesis.^ To facilitate the cloning of ERCC4 gene and, in general, the cloning of genes from any defined regions of the human genome, a method was developed for the direct isolation of human transcribed genes ffom somatic cell hybrids. cDNA was prepared from human x rodent hybrid by using consensus 5$\sp\prime$ splice site sequences as primers. These primers were designed to select immature, unspliced messenger RNA (still retaining species specific repeat sequences) as templates. Screening of a derived cDNA library for human repeat sequences resulted in the isolation of human clones at the anticipated frequency with characteristics expected of exons of transcribed human genes. The usefulness of the splice site specific primers was analyzed and the cDNA synthesis conditions with these primers were optimized. The procedure was shown to be sensitive enough to clone weakly expressed genes. Studying the expression of the represented genes with the isolated clones was shown to be feasible. Such regional specific human gene fragments will be very valuable for many human genetic studies such as the search of inherited disease genes and the construction of a cDNA map of the human genome. ^

The molecular cloning and characterization of human heparanase cDNA and the immunochemical localization of heparanase in metastatic melanomas

Heparanase, an endo-$\beta$-D-glucuronidase, has been associated with melanoma metastasis. Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a M$\sb{\rm r}\sim 97,000$ protein upon SDS-polyacrylamide gel electrophoresis of mouse melanoma and human melanoma cell lysates. In an indirect immunocytochemical study, metastatic human A375-SM and mouse B16-BL6 melanoma cells were stained with the anti-heparanase antibodies. Heparanase antigen was localized in the cytoplasm of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells. Immunohistochemical staining of frozen sections from syngeneic mouse organs containing micrometastases of B16-BL6 melanoma demonstrated heparanase localized in metastatic melanoma cells, but not in adjacent normal tissues. Similar studies using frozen sections of malignant melanomas resected from patients indicated that heparanase is localized in invading melanoma cells, but not in adjacent connective tissues.^ Monoclonal antibodies directed against murine heparanase were developed and characterized. Monoclonal antibody 10E5, an IgM, precipitated and inhibitated the enzymatic activity of heparanase. A 2.6 kb cDNA was isolated from a human melanoma $\lambda$gt11 cDNA library using the monoclonal antibody 10E5. Heparan sulfate cleavage activity was detected in the lysogen lysates from E. Coli Y1089 infected with the $\lambda$gt11 cDNA and this activity was inhibited in the presence of 10-fold excess of heparin, a potent inhibitor of heparanase. The nucleotide sequence of the cDNA was determined and insignificant homology was found with the gene sequences currently known. The cDNA hybridized to a 3.2-3.4 kb mRNA in human A375 melanoma, WI-38 fibroblast, and THP-1 leukemia cells using Northern blots.^ Heparanase expression was examined using Western and Northern blots. In comparison to human A375-P melanoma cells, the quantity of 97,000 protein recognized by the polyclonal anti-heparanase antibodies doubled in the metastatic variant A375-SM cells and the quantity of 3.2-3.4 kb mRNA doubled in A375MetMix, a metastatic variant similar to A375-SM cells. In B16 murine melanoma cell, the intensity of the 97,000 protein increased more than 2 times comparing with B16-F1 cells. The extent in the increase of the protein and the mRNA levels is comparable to the change of heparanase activity observed in those cells.^ In summary, the studies suggest that (a) the N-terminus of the heparanase molecule in mouse and human is antigenically related; (b) heparanase antigens are localized at the cell surface and in the cytoplasm of metastatic human and mouse melanoma cells; (c) heparanase antigens are localized in invasive and metastatic murine and human melanomas in vivo, but not in adjacent normal tissues; (d) heparanase molecule appeared to be differentially expressed at the transcriptional as well as at the translational level; and (e) the size of human heparanase mRNA is 3.2-3.4 kilobase. ^

cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^

Cloning and characterization of the mouse ret finger protein (rfp), a B box zinc finger protein

An important question in biology is to understand the role of specific gene products in regulating embryogenesis and cellular differentiation. Many of the regulatory proteins possess specific motifs, such as the homeodomain, basic helix-loop-helix structure, zinc finger, and leucine zipper. These sequence motifs participate in specific protein-DNA, protein-RNA, and protein-protein interactions, and are important for the function of these regulatory proteins.^ The human rfp (ret finger protein) belongs to a novel zinc finger protein family, the B box zinc finger family. Most of the B box proteins, including rfp, have a conserved tripartite motif, consisting of two novel zinc fingers (the RING finger and the B box) and a coiled-coil domain. Interestingly, a fusion protein between the tripartite motif of rfp and the tyrosine kinase domain of c-ret has transforming activity. In this study, we examined the expression of rfp during mouse development, and characterized the role of the tripartite motif in rfp function.^ We cloned the mouse rfp cDNA, which shares a 98.4% homology with the human sequence at amino acid level. Such strikingly high degree of homology indicates the high evolutionary pressure on the conservation of the sequence, suggesting that rfp may have an important function. Using the somatic cell hybrid system, we assigned the rfp gene to mouse chromosome 13 and human chromosome 6. Rfp transcripts and protein were ubiquitous in day 10.5-13.5 mouse embryos; however, they were restricted in adult mice, with the highest level of expression in the testis. Rfp expression in the testis is detected only in late pachytene spermatocytes and round spermatids. In both embryos and spermatogenic cells, rfp protein was distributed within cell nuclei in a punctate pattern, similar to the PODs (PML oncogenic domains) observed with another B box protein, PML. In cultured mammalian cells, we found that rfp was indeed co-localized to the PODs with PML. Using the yeast two-hybrid system, we showed that the rfp could specifically interact with PML, and that the interaction was dependent on the distal portion of the rfp coiled-coil domain.^ We also showed that rfp could form homodimers, and both the B box and coiled-coil domain were required for proper dimerization. It seems that the proximal portion of the coiled-coil domain provides the interacting interface, while the B box zinc finger orients the coil and maintains the correct structure of the whole molecule. Our data are consistent with the zinc-binding property and structural analysis of the B box. The RING finger seems to be involved in rfp nuclear localization through interaction with other proteins. We believe that homodimerization and interaction with PML are important for the normal interaction of rfp during development and differentiation. In addition, rfp homodimerization may also be essential for the oncogenic activation of the rfp-ret fusion protein. ^

Gene transfer by viral vectors

To initiate our clinical trial for chemotherapy protection, I established the retroviral vector system for human MDR1 cDNA gene transfer. The human MDR1 cDNA continued to be expressed in the transduced bone marrow cells after four cohorts of serial transplants, 17 months after the initial transduction and transplant. In addition, we used this retroviral vector pVMDR1 to transduce human bone marrow and peripheral blood CD34$\sp+$ cells on stromal monolayer in the presence of hematopoietic growth factors. These data suggest that the retroviral vector pVMDR1 could modify hematopoietic precursor cells with a capacity for long-term self renewal. Thus, it may be possible to use the MDR1 retroviruses to confer chemotherapeutic protection on human normal hematopoietic precursor cells of ovarian and breast cancer patients in whom high doses of MDR drugs may be required to control the diseases.^ Another promising vector system is recombinant adeno-associated virus (rAAV) vector. An impediment to use rAAV vectors is that production of rAAV vectors for clinical use is extremely cumbersome and labor intensive. First I set up the rAAV vector system in our laboratory and then, I focused on studies related to the production of rAAV vectors for clinical use. By using a self-inactivating retroviral vector carrying a selection marker under the control of the CMV immediate early promoter and an AAV genome with the deletion of both ITRs, I have developed either a transient or a stable method to produce rAAV vectors. These methods involve infection only and can generate high-titer rAAV vectors (up to 2 x 10$\sp5$ cfu/ml of CVL) with much less work.^ Although recombinant adenoviral vectors hardly infect early hematopoietic precursor cells lacking $\alpha\sb v\beta\sb5$ or $\alpha\sb v\beta\sb3$ integrin on their surface, but efficiently infect other cells, we can use these properties of adenoviral vectors for bone marrow purging as well as for development of new viral vectors such as pseudotyped retroviral vectors and rAAV vectors. Replacement of self-inactivating retroviral vectors by recombinant adenoviral vectors will facilitate the above strategies for production of new viral vectors. In order to accomplish these goals, I developed a new method which is much more efficient than the current methods to construct adenoviral vectors. This method involves a cosmid vector system which is utilized to construct the full-length recombinant adenoviral vectors in vitro.^ First, I developed an efficient and flexible method for in vitro construction of the full-length recombinant adenoviral vectors in the cosmid vector system by use of a three-DNA fragment ligation. Then, this system was improved by use of a two-DNA fragment ligation. The cloning capacity of recombinant adenoviral vectors constructed by this method to develop recombinant adenoviral vectors depends on the efficiency of transfection only. No homologous recombination is required for development of infectious adenoviral vectors. Thus, the efficiency of generating the recombinant adenoviral vectors by the cosmid method reported here was much higher than that by the in vitro direct ligation method or the in vivo homologous recombination method reported before. This method of the in vitro construction of recombinant adenoviral vectors in the cosmid vector system may facilitate the development of adenoviral vector for human gene therapy. (Abstract shortened by UMI.) ^