Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reihardtii and Volvox carteri: phylogenetic origins and recent insertion of introns.

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble "proto-splice" sequences in the Chlamydomonas gene.

Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1.

Genomic complementation of the high-CO2-requiring mutant ca-1-12-1C of Chlamydomonas reinhardtii was achieved by transformation with DNA pools from an indexed cosmid library of wild-type genomic DNA. Transformation of mutant cells with cosmid DNA from two microtiter plates in the library produced colonies that grew phototrophically at atmospheric CO2 levels. Transformations with cosmid DNA from each of the rows and files of the two plates pinpointed one well in each plate with a cosmid bearing the targeted gene. Sequencing of cosmid subclones revealed a gene encoding a recently identified C. reinhardtii chloroplast carbonic anhydrase (CAH3). Transformations with chimeric constructs combining different portions of the wild-type and mutant genes indicated the presence of a mutation in the 5'-half of the gene. Comparison of mutant and wild-type gene sequences in this region revealed a G-to-A substitution in the mutant gene, which produced a nonsense codon. The data presented demonstrate that the carbonic anhydrase produced from the CAH3 gene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii and that genomic complementation can be a facile and efficient means for isolating genes associated with defects affecting photosynthesis and other physiological processes in this eukaryotic green alga.

Characterization and application of soybean YACs to molecular cytogenetics.

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.

Physical mapping of a region in the soybean (Glycine max) genome containing duplicated sequences.

Pulsed-field gel electrophoresis (PFGE) was used to study a cluster of molecular markers in the soybean genome. There were 550 kb per centimorgan (cM) in the cluster, which is close to the calculated average for the whole genome. The analysis was complicated by the presence of duplicated sequences, and some ambiguities arising from this were resolved by using second-dimension conventional electrophoresis to relate physical maps to the RFLP map of soybean. The results show that there is a high degree of conservation of 'rare cutter' sites between homoeologous regions. Finally, PFGE can confirm physical linkage of monomorphic copies of markers, which can aid in the study and comparison of homoeologous regions that are invisible to RFLP analysis.

DAF-amplified fragments can be used as markers for DNA from pulse field gels.

PCR-type amplification of genomic sequences using short, single and arbitrarily chosen primers (by DNA amplification fingerprinting [DAF]) was developed in our laboratory. Here we show that it is possible to produce specific fingerprints for subgenomic DNA fragments embedded in agarose, including fractions from conventional and pulse field gels. Fractions of restricted genomic DNA differing in mobility by only 2-3 mm gave one to three different products with different single primers. Yeast chromosome specific markers were easily produced and cloned. We also used DAF to generate specific amplification products of DNA cloned into a yeast artificial chromosome. These products were readily cloned into a plasmid vector.

The influence of temperature on muscle velocity and sustained performance in swimming carp.

The aim of this study was to evaluate how fish locomote at different muscle temperatures. Sarcomere length excursion and muscle shortening velocity, V, were determined from high-speed motion pictures of carp, Cyprinus carpio (11-14 cm), swimming steadily at various sustained speeds at 10, 15 and 20 degrees C. In the middle and posterior regions of the carp, sarcomeres of the lateral red muscle underwent cyclical excursions of 0.31 microns, centered around the resting length of 2.06 microns (i.e. from 1.91 to 2.22 microns). The amplitudes of the sarcomere length excursions were essentially independent of swimming speed and temperature. As tail-beat frequency increased linearly with swimming speed regardless of temperature, the sarcomeres underwent the same length changes in a shorter time. Thus, V increased in a linear and temperature-independent manner with swimming speed. Neither temperature nor swimming speed had an influence on tail-beat amplitude or tail height. Our findings indicate that muscle fibres are used only over a narrow, temperature-independent range of V/Vmax (0.17-0.36) where power and efficiency are maximal. Carp start to recruit their white muscles at swimming speeds where the red muscle V/Vmax becomes too high (and thus power output declines). When the V/Vmax of the active muscle falls too low during steady swimming, carp switch to 'burst-and-coast' swimming, apparently to keep V/Vmax high. Because Vmax (maximum velocity of shortening) of carp red muscle has a Q10 of 1.63, the transition speeds between swimming styles are lower at lower temperatures. Thus, carp recruit their white anaerobic muscle at a lower swimming speed at lower temperatures (verified by electromyography), resulting in a lower maximum sustainable swimming speed. The present findings also indicate that, to generate the same total force and power to swim at a given speed, carp at 10 degrees C must recruit about 50% greater fibre cross-sectional area than they do at 20 degrees C.

Why animals have different muscle fibre types.

Animals have different muscle fibre types: slow fibres with a low maximum velocity of shortening (Vmax) and fast fibres with a high Vmax. An advantage conferred by the use of different fibre types during locomotion has been proposed solely on the basis of their in vitro properties. Isolated muscle experiments show that force generation, mechanical power production and efficiency are all functions of V/Vmax, where V is the velocity of muscle shortening. But it is not known whether animals actually use the different fibres at shortening velocities that are optimal for mechanical power production and efficiency. Here we compare the V of muscle fibres during locomotion with their Vmax. This comparison shows that during slow locomotion, the slow fibres shorten at a velocity that gives peak mechanical power and efficiency and the fast fibres shorten at their optimal velocity when powering maximal movements. Our results also show that maximal movements are impossible without fast fibres because the slow ones cannot shorten rapidly enough.