Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene

We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.     

Cardiovascular effects of training for a marathon run in unfit middle aged men.

The effects of a 30 week exercise programme on serum lipid values, blood pressure, and cardiac function were assessed in a group of sedentary men aged 35-50 training for their first marathon. Mean serum cholesterol concentration (n = 33) fell by 12% from 6.54 (SE 0.18) to 5.76 (0.15) mmol/l (mean fall 0.78 mmol/l; 95% confidence interval 0.52 to 1.04 mmol/l), serum triglyceride concentration (n = 33) by 22% from 1.56 (0.17) to 1.21 (0.09) mmol/l (mean fall 0.34 mmol/l; 95% confidence interval 0.12 to 0.56 mmol/l), and mean blood pressure (n = 27) by 10% from 102 (2) to 92 (2) mm Hg (mean fall 10 mm Hg; 95% confidence interval 7 to 13 mm Hg). These changes were not explained by changes in body composition. Peak exercise left ventricular end diastolic volume (n = 16) increased with training; as a result of this and an increased exercise left ventricular ejection fraction peak exercise cardiac output increased from 19.9 (1.2) to 23.1 (3.0) l/min (mean rise 3.2 l/min; 95% confidence interval 1.5 to 5.0 l/min). Maximum oxygen consumption increased from 33.9 (1.6) to 39.0 (1.3) ml/kg/min (mean rise 5.0 ml/kg/min; 95% confidence interval 1.8 to 8.2 ml/kg/min). This study showed favourable effects on coronary risk factors and cardiac function and supports the place of regular exercise in coronary prevention programmes.     

Constancy of synaptic ribbon numbers in the retina of the arctic chary,Salvelinus alpinus

Synopsis At high latitudes, such as in Iceland, the daily photoperiod varies from almost continuous darkness in winter to virtually constant light in summer. Previous studies of detailed retinal structure in vertebrates have shown significant daily and annual effects of photoperiod. We sampled arctic charr in Iceland during the summer, including fish that were both light- and dark-adapted, during both day and night. We observed retinomotor responses characteristic of light- and dark-adaptation, but found no difference in the number of synaptic ribbons in the retina. The morpho-physiological changes, appearing as retinomotor responses, are thus not expressed at the synaptic level.     

Characterization of the cDNA coding for mouse plasminogen and localization of the gene to mouse chromosome 17

A full-length cDNA coding for mouse plasminogen has been isolated and characterized. The cDNA is 2720 bp in length (excluding the poly(A) tail) and contains a 24-bp 5' noncoding region, an open reading frame of 2436 bp, and a 3' noncoding region of 257 bp. The open reading frame codes for 812 amino acids and includes a signal peptide that is likely 19 amino acids in length and the mature protein of 793 amino acids. The calculated Mr of mouse plasminogen is 88,706 excluding carbohydrate. There are two potential N-linked carbohydrate addition sites; one of which is glycosylated in human, bovine, and porcine plasminogens. Mouse plasminogen was found to contain two additional amino acids compared to the human protein. In addition, mouse and human plasminogens were found to be 79 and 76% identical at the protein and DNA levels, respectively. Analysis of the segregation of two allelic forms, Plgb and Plgd, of plasminogen DNA in three sets of recombinant inbred strains has allowed the localization of the mouse plasminogen gene to the proximal end of mouse chromosome 17 within the t complex and close to the locus D17Rp17. The Plg gene is deleted in the semidominant deletion mutant, hair-pintail (Thp).     

Effects of Cellulolytic Ruminal Bacteria and of Cell Extracts on Germination of Euonymus americanus L. Seeds

In past attempts, the experimental germination of the seeds of Euonymus americanus L. in vitro has had little success. However, treatment of seeds with ruminal fluid containing viable microflora has been successful in stimulating germination. In the presence of the cellulolytic ruminal bacterium, Clostridium cellobioparum ATCC 15832, seeds of E. americanus were stimulated to germinate. Subsequent studies were designed to determine whether the bacterium synthesized a cellulolytic enzyme responsible for initiating germination. The cell-free endocellulase from C. cellobioparum induced germination of the seeds. To support the hypothesis that the endocellulase from C. cellobioparum was responsible for triggering germination, a 1,4-beta-d-glucan glucanohydrolase (EC 3.2.1.4) from Penicillum funiculosum was used to treat the seeds. In addition, no germination was obtained from seeds treated with a commercial exocellulase enzyme. Also, Ruminococcus flavefaciens FD-1 was found to initiate germination of E. americanus seeds. Thus, cellulase activity is indicated in the degradation of the testa of the seed, allowing imbibition and germination.     

Magna-Site tissue expander: an innovation for injection site location

The Magna-Site tissue expander simplifies the use of tissue expanders by facilitating the location of the injection port, thereby reducing the risks associated with faulty injection placement in the past. The injection port is smooth except for the innocuous palpation bumps, drastically reducing the problems of erosion at the injection port site encountered in the past. No drawbacks were encountered with the use of this expander relative to the use of other currently marketed expanders. The new product is available at a cost similar to expanders currently available. It is felt by the authors that this expander represents the next logical step in the evolution of this technology. Just as Dr. Cohen thought, "It will be exciting to see what new devices are created to further enhance the concept of tissue expansion."