Grapevines produce non-climacteric fruit that exhibit a double sigmoidal pattern of growth. Ripening occurs during the second growth phase when grapes change colour, start to soften, accumulate reducing sugars, metabolise organic acids and synthesise flavour compounds. Unlike many other fruit, grapes ripen while the berries are still expanding, and as with most non-climacteric fruit, ripening does not appear to be controlled by ethylene. Sugars and amino acids that accumulate in grapes during ripening are imported via the phloem, while many secondary metabolites are synthesised within the berry itself. Grapes import sucrose but accumulate hexoses. Conversion of sucrose to hexoses is most likely catalysed by invertase. cDNAs encoding vacuolar invertases have been isolated from grape berries. Expression of these genes and an increase in invertase activity occur before veraison, so it seems unlikely that synthesis of this enzyme is a controlling factor for sugar accumulation during ripening. Proteins that transport sugars into the berry vacuole may regulate sugar accumulation, and cDNAs encoding both sucrose and hexose transporters have been isolated from ripening grape berries. Determination of the role of these transporters may reveal the pathway of sugar accumulation in grapes. Anthocyanins are only synthesised in the skin of red grapes after veraison. Analysis of the patterns of expression of genes in the flavonoid pathway has shown that there is a dramatic increase in expression of many of these genes in skin cells at veraison. Expression of the gene encoding a glycosyl transferase involved in the lasts steps of anthocyanin synthesis was absolutely correlated with anthocyanin synthesis and may explain the lack of anthocyanin synthesis in white grapes and in the flesh of most red grapes. We infer that the synthesis of anthocyanins is regulated at the transcription level and is likely to be controlled by regulatory genes. Softening of fruit generally results from changes in the properties of cell walls. Analysis of the cell walls of grapes during ripening suggests that there are no dramatic changes in polysaccharide composition but modification of specific components may contribute to softening. A number of proteins are newly synthesised in grapes during ripening and several of these proteins have now been identified. The most abundant are pathogenesis-related (PR) proteins, including chitinases and thaumatin-like proteins. Expression of genes encoding a number of PR proteins increased dramatically in grapes during ripening. It is not clear what role the PR proteins play during ripening but they may provide resistance to pathogens. Differential screening of a post-veraison grape berry cDNA library has also identified ripening-related genes, some of which encode proline-rich cell wall proteins. Other grape ripening-related genes have homologues that are induced by stress in other plants. These studies indicate that a dramatic change in gene expression occurs in grape berries at veraison and suggest that ripening involves a coordinated increase in transcription of a number of different genes.
Usher syndrome 1C (USH1C) is a congenital condition manifesting profound hearing loss, the absence of vestibular function, and eventual retinal degeneration. The USH1C locus has been mapped genetically to a 2- to 3-cM interval in 11p14–15.1 between D11S899 and D11S861. In an effort to identify the USH1C disease gene we have isolated the region between these markers in yeast artificial chromosomes (YACs) using a combination of STS content mapping and Alu –PCR hybridization. The YAC contig is ∼3.5 Mb and has located several other loci within this interval, resulting in the order CEN-LDHA-SAA1-TPH-D11S1310-(D11S1888/KCNC1)-MYOD1-D11S902D11S921-D11S1890-TEL. Subsequent haplotyping and homozygosity analysis refined the location of the disease gene to a 400-kb interval between D11S902 and D11S1890 with all affected individuals being homozygous for the internal marker D11S921. To facilitate gene identification, the critical region has been converted into P1 artificial chromosome (PAC) clones using sequence-tagged sites (STSs) mapped to the YAC contig, Alu –PCR products generated from the YACs, and PAC end probes. A contig of >50 PAC clones has been assembled between D11S1310 and D11S1890, confirming the order of markers used in haplotyping. Three PAC clones representing nearly two-thirds of the USH1C critical region have been sequenced. PowerBLAST analysis identified six clusters of expressed sequence tags (ESTs), two known genes ( BIR,SUR1 ) mapped previously to this region, and a previously characterized but unmapped gene NEFA (D N A binding/ EF hand/ a cidic amino-acid-rich). GRAIL analysis identified 11 CpG islands and 73 exons of excellent quality. These data allowed the construction of a transcription map for the USH1C critical region, consisting of three known genes and six or more novel transcripts. Based on their map location, these loci represent candidate disease loci for USH1C. The NEFA gene was assessed as the USH1C locus by the sequencing of an amplified NEFA cDNA from an USH1C patient; however, no mutations were detected. [The sequence data described in this paper have been submitted to GenBank under accession numbers AC000406 – AC000407 .]
Transforming growth factor β1 (TGF-β1) has a potent profibrotic function and is central to signaling cascades involved in interstitial fibrosis, which plays a critical role in the pathobiology of cardiomyopathy and contributes to diastolic and systolic dysfunction. In addition, fibrotic remodeling is responsible for generation of re-entry circuits that promote arrhythmias (Bujak and Frangogiannis 2007 Cardiovasc. Res. 74, 184–195). Due to the small size of the heart, functional electrophysiology of transgenic mice is problematic. Large transgenic animal models have the potential to offer insights into conduction heterogeneity associated with fibrosis and the role of fibrosis in cardiovascular diseases. The goal of this study was to generate transgenic goats overexpressing an active form of TGFβ-1 under control of the cardiac-specific α-myosin heavy chain promoter (α-MHC). A pcDNA3.1DV5-MHC-TGF-β1cysser vector was constructed by subcloning the MHC-TGF-β1 fragment from the plasmid pUC-BM20-MHC-TGF-β1 (Nakajima et al. 2000 Circ. Res. 86, 571–579) into the pcDNA3.1D V5 vector. The Neon transfection system was used to electroporate primary goat fetal fibroblasts. After G418 selection and PCR screening, transgenic cells were used for SCNT. Oocytes were collected by slicing ovaries from an abattoir and matured in vitro in an incubator with 5% CO2 in air. Cumulus cells were removed at 21 to 23 h postmaturation. Oocytes were enucleated by aspirating the first polar body and nearby cytoplasm by micromanipulation in Hepes-buffered SOF medium with 10 μg of cytochalasin B mL. Transgenic somatic cells were individually inserted into the perivitelline space and fused with enucleated oocytes using double electrical pulses of 1.8 kV cm (40 μs each). Reconstructed embryos were activated by ionomycin (5 min) and DMAP and cycloheximide (CHX) treatments. Cloned embryos were cultured in G1 medium for 12 to 60 h in vitro and then transferred into synchronized recipient females. Pregnancy was examined by ultrasonography on day 30 post-transfer. A total of 246 cloned embryos were transferred into 14 recipients that resulted in production of 7 kids. The pregnancy rate was higher in the group cultured for 12 h compared with those cultured 36 to 60 h [44.4% (n = 9) v. 20% (n = 5)]. The kidding rates per embryo transferred of these 2 groups were 3.8% (n = 156) and 1.1% (n = 90), respectively. The PCR results confirmed that all the clones were transgenic. Phenotype characterization [e.g. gene expression, electrocardiogram (ECG), and magnetic resonance imaging (MRI)] is underway. We demonstrated successful production of transgenic goat via SCNT. To our knowledge, this is the first transgenic goat model produced for cardiovascular research. This work was supported by the Utah Science Technology and Research Initiative, Utah Multidisciplinary Arrhythmia Consortium.
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