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Purpose: To investigate single nucleotide polymorphisms (SNPs) and trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene in a large cohort of German patients with Fuchs endothelial corneal dystrophy (FECD)., Methods: Genomic DNA was obtained from 398 patients with FECD and from 58 non-FECD controls. Thirty-seven previously reported SNPs were evaluated by genotyping. The 398 FECD samples were analyzed for TNR expansions by short tandem repeat assays and Southern blotting. The possible associations between the TNR length and clinical parameters (age, sex, visual acuity, and central corneal thickness) were analyzed in 132 patients., Results: The SNPs in COL8A2, TCF8, LOXHD1, and AGBL1 showed no heterogeneity in 36 cases, although SLCA411 showed 3 nonsense mutations. SNPs were detected for TCF4 (rs613872, rs2123392, rs17089887, rs1452787, and rs1348047), but only rs613872 showed a significant association with FECD (P = 9.93 × 10). Overall, 315/398 (79%) patients harbored TNR lengths >50, whereas no non-FECD controls harbored TNR lengths >50. The TCF4 SNP rs613872 genotype was TT: 39 (67%), TG: 18 (31%), and GG: 1 (2%) in non-FECD controls; TT: 39 (47%), TG: 38 (46%), and GG: 6 (7%) in FECD cases harboring TNR <50; and TT: 23 (8%), TG: 224 (79%), and GG: 38 (13%) in FECD cases harboring TNR >50 (P = 2.93 × 10). No significant association was detected between the TNR length and clinical parameters., Conclusions: Our large German cohort demonstrated a significant association between the risk allele G in rs613872 and FECD, irrespective of TNR expansion, although this risk allele was more frequent in FECD cases with TNR expansion than without.

Purpose: CTG trinucleotide repeat (TNR) expansion is frequently found in transcription factor 4 (TCF4) in Fuchs' endothelial corneal dystrophy (FECD), though the effect of TNR expansion on FECD pathophysiology remains unclear. The purpose of this study was to evaluate the effect of TNR expansion on TCF4 expression in corneal endothelium of patients with FECD., Methods: Peripheral blood DNA and Descemet membrane with corneal endothelium were obtained from 203 German patients with FECD. The CTG TNR repeat length in TCF4 was determined by short tandem repeat (STR) assays and Southern blotting using genomic DNA. Genotyping of rs613872 in TCF4 was performed by PCR. TCF4 mRNA levels in corneal endothelium were evaluated by quantitative PCR using three different probes. Control corneal endothelial samples were obtained from 35 non-FECD subjects., Results: The STR assay and Southern blotting showed that 162 of the 203 patients with FECD (80%) harbored CTG trinucleotide repeat lengths larger than 50. Quantitative PCR using all three probes demonstrated that TCF4 mRNA is significantly upregulated in the corneal endothelium of patients with FECD, regardless of the presence of TNR expansion. However, the length of the TNR tended to show a positive correlation with TCF4 expression level. No correlation was shown between the genotype of TCF4 SNP, rs613872, and the level of TCF4 expression., Conclusions: Our findings showed that TCF4 mRNA is upregulated in the corneal endothelium of patients with FECD. Further studies on the effects of TCF4 upregulation on corneal endothelial cell function will aid in understanding the pathophysiology of FECD.

In situ hybridization (ISH) was performed using oral biopsies from patients with paracoccidioidomycosis and guinea pig testes inoculated with a culture of Paracoccidioides brasiliensis isolated from soil, employing both a 14 base-pair specific oligoprobe (ACT CCC CCG TGG TC) and its complementary sequence. When combining ISH with the Gridley stain which detects fungal cell walls, about 2-3% of the fungal cells present in the tissues were labelled. When the complementary probe was used, labelling was higher, reaching the 3% level.

Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.

The purpose of the present study was to examine the effects of acute (3 days) unilateral diaphragm denervation (DNV) on 1) levels of alpha 1(I) and alpha 1(III) procollagen mRNA; 2) collagen concentration [hydroxyproline (HYP)]; 3) amount of the nonreducible collagen cross-link hydroxylysylpyridinoline (HP); and 4) the passive force-length relationship of the muscle. The levels of alpha 1(I) and alpha 1(III) procollagen mRNA, HYP concentration, and amount of HP were measured in muscle segments from the midcostal region of DNV and intact (INT) hemidiaphragms of adult male Fischer 344 rats (250-300 g). The in vitro passive force-length relationship of DNV and INT hemidiaphragm was determined by lengthening and shortening the diaphragm muscle segments from 85 to 115% of optimal length at a constant velocity (0.6 optimal length/s). Three days after DNV, the level of alpha 1(I) procollagen mRNA was increased over 15-fold in the DNV hemidiaphragm compared with INT (P < 0.05), whereas the level of alpha 1(III) procollagen mRNA was increased by approximately sixfold in the DNV hemidiaphragm compared with INT (P < 0.05). Collagen (HYP) concentration did not differ between groups, averaging 8.7 and 8.9 micrograms/mg dry wt for the DNV and INT hemidiaphragms, respectively. In addition, there was no difference in the amount of the mature nonreducible collagen cross-link HP between the DNV and INT hemidiaphragms (0.66 vs. 0.76 mole HP/mole collagen, respectively). The amount of passive force developed during lengthening did not differ between DNV and INT hemidiaphragms. These data indicate that acute DNV of the hemidiaphragm is associated with an increase in the mRNA level of the two principal fibrillar collagen phenotypes in skeletal muscle. However, despite extensive muscle remodeling, the passive force-length relationship of the DNV hemidiaphragm is unaffected compared with the INT muscle.

Plasmid F replication is controlled by a plasmid-specified Rep protein with both autorepressor and initiator functions. The mechanism by which these two functions of a Rep protein are balanced to achieve stable replication is unknown; however, we speculated in prior work that Rep protein modification could be involved. We report here that naturally proteolyzed F RepE protein has been detected and characterized. The processed molecule lost the first 17 N-terminal aminoacyl residues and initiator function but acquired increased specific DNA-binding affinity in the presence of Escherichia coli chromosomal DNA. When supplied in trans, the altered protein acts as an incompatibility substance and eliminates maintenance of F'lac. These findings indicate that protein processing has the potential to contribute to the overall control of DNA replication.

We have developed a one-step process for constructing synthetic genes. Four adjacent oligonucleotides 17-100 bases in length having short overlaps of 15-17 bases are used as primers in a PCR mixture. The quantity of the two internal primers is highly limited, and the resultant reaction causes an asymmetric single-stranded amplification of the two halves of the total sequence due to an excess of the two flanking primers. In subsequent PCR cycles, these dual asymmetrically amplified fragments, which overlap each other, yield a double-stranded, full-length product.

Chromosomal mutants were isolated in which, for several small plasmids, there was an increased amount of either covalently closed circular plasmid DNA or total plasmid DNA or both. The mutations were mapped to recD, which has been shown to affect exonuclease V activity and a variety of plasmid maintenance and replication functions. Our results suggest that rolling-circle plasmid replication can occur in recD mutants and that site-specific recombination can resolve the resulting linear multimers into covalently closed circular plasmid forms.