Cytogenetic analyses of eight species in the genus <Emphasis Type="Italic">Leptodactylus</Emphasis> Fitzinger, 1843 (Amphibia,Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations

Background

The karyotypes of Leptodactylus species usually consist of 22 bi-armed chromosomes, but morphological variations in some chromosomes and even differences in the 2n have been reported. To better understand the mechanisms responsible for these differences, eight species were analysed using classical and molecular cytogenetic techniques, including replication banding with BrdU incorporation.

Results

Distinct chromosome numbers were found: 2n = 22 in Leptodactylus chaquensis, L. labyrinthicus, L. pentadactylus, L. petersii, L. podicipinus, and L. rhodomystax; 2n = 20 in Leptodactylus sp. (aff. podicipinus); and 2n = 24 in L. marmoratus. Among the species with 2n = 22, only three had the same basic karyotype. Leptodactylus pentadactylus presented multiple translocations, L. petersii displayed chromosome morphological discrepancy, and L. podicipinus had four pairs of telocentric chromosomes. Replication banding was crucial for characterising this variability and for explaining the reduced 2n in Leptodactylus sp. (aff. podicipinus). Leptodactylus marmoratus had few chromosomes with a similar banding patterns to the 2n = 22 karyotypes. The majority of the species presented a single NOR-bearing pair, which was confirmed using Ag-impregnation and FISH with an rDNA probe. In general, the NOR-bearing chromosomes corresponded to chromosome 8, but NORs were found on chromosome 3 or 4 in some species. Leptodactylus marmoratus had NORs on chromosome pairs 6 and 8. The data from C-banding, fluorochrome staining, and FISH using the telomeric probe helped in characterising the repetitive sequences. Even though hybridisation did occur on the chromosome ends, telomere-like repetitive sequences outside of the telomere region were identified. Metaphase I cells from L. pentadactylus confirmed its complex karyotype constitution because 12 chromosomes appeared as ring-shaped chain in addition to five bivalents.

Conclusions

Species of Leptodactylus exhibited both major and minor karyotypic differences which were identified by classical and molecular cytogenetic techniques. Replication banding, which is a unique procedure that has been used to obtain longitudinal multiple band patterns in amphibian chromosomes, allowed us to outline the general mechanisms responsible for these karyotype differences. The findings also suggested that L. marmoratus, which was formerly included in the genus Adenomera, may have undergone great chromosomal repatterning.

     

Prokaryotic gene finding based on physicochemical characteristics of codons calculated from molecular dynamics simulations

An ab initio model for gene prediction in prokaryotic genomes is proposed based on physicochemical characteristics of codons calculated from molecular dynamics (MD) simulations. The model requires a specification of three calculated quantities for each codon: the double-helical trinucleotide base pairing energy, the base pair stacking energy, and an index of the propensity of a codon for protein-nucleic acid interactions. The base pairing and stacking energies for each codon are obtained from recently reported MD simulations on all unique tetranucleotide steps, and the third parameter is assigned based on the conjugate rule previously proposed to account for the wobble hypothesis with respect to degeneracies in the genetic code. The third interaction propensity parameter values correlate well with ab initio MD calculated solvation energies and flexibility of codon sequences as well as codon usage in genes and amino acid composition frequencies in ∼175,000 protein sequences in the Swissprot database. Assignment of these three parameters for each codon enables the calculation of the magnitude and orientation of a cumulative three-dimensional vector for a DNA sequence of any length in each of the six genomic reading frames. Analysis of 372 genomes comprising ∼350,000 genes shows that the orientations of the gene and nongene vectors are well differentiated and make a clear distinction feasible between genic and nongenic sequences at a level equivalent to or better than currently available knowledge-based models trained on the basis of empirical data, presenting a strong support for the possibility of a unique and useful physicochemical characterization of DNA sequences from codons to genomes.     

Use of metabolic stimulators and inhibitors for enhanced production of beta-carotene and lycopene by Blakeslea trispora NRRL 2895 and 2896

This work investigates the potential of metabolic stimulators, firstly to enhance the production of beta-carotene, and later use of inhibitors of lycopene cyclase so as to accumulate lycopene in the fermentation medium. Various non-ionic surfactants, natural oils, stimulators such as amino-acids, tricarboxylic acid cycle (TCA) intermediates, vitamin A and antibiotics were investigated for improved production of beta-carotene using the zygomycete fungus Blakeslea trispora. Span 20 at 0.2% increased the beta-carotene production from 139 mg/l to 318 mg/l. Examination of the mycelial morphology of the B. trispora with span 20 showed a shorter mycelial length, which allowed a well-dispersed growth of B. trispora. Supplementation of the medium with 1000 ppm vitamin A acetate gave highest concentration of beta-carotene (830+/-6 mg/l). Several chemical inhibitors such as imidazole, pyridine, triethylamine, piperidine, and nicotinic acid were then evaluated to block the biosynthesis at lycopene. Piperidine at 500 ppm gave a 7.76-fold improvement, and produced high titers of lycopene (775+/-5 mg/l) in a medium supplemented with vitamin A acetate.     

The influence of p,p'-DDT, alpha-chlordane, heptachlor and endrin on hepatic and renal carbohydrate metabolism and cyclic AMP-adenyl cyclase system

Administration of an acute oral dose of p,p′-DDT (600 mg/kg), α-chlordane (200 mg/kg), heptachlor (200 mg/kg) and endrin (50 mg/kg) produced a significant rise in the concentration of serum glucose and urea and a lowering of hepatic glycogen. In addition, treatment with either of these insecticides significantly increased the activities of hepatic and renal pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-diphosphatase and glucose 6-phosphatase, the four enzymes which play a key, rate-limiting role in the process of gluconeogenesis. Treatment with p,p′-DDT, α-chlordane, heptachlor or endrin proved equally effective in elevating the levels of endogenous cyclic AMP and augmenting the activity of basal- and fluoride-stimulated forms of adenyl cyclase in both tissues. Whereas renal phosphodiesterase was decreased slightly by p,p′-DDT, the activity of this cyclic AMP-degrading enzyme remained unaltered following the administration of other pesticides. Our data indicate that the pesticide-induced alterations in carbohydrate metabolism of liver and kidney may be associated with an enhanced ability of these organs to synthesize cyclic AMP.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Optimisation of conditions of synthesis of oxidised starch from corn and amaranth for use in film-forming applications

Gum arabic is used as an encapsulating agent because of its film-forming ability. However, India has to import gum arabic for its domestic requirement. Oxidised starch has been reported as a substitute for gum arabic but no data are reported on the exact conditions of oxidation of starch or the analytical indicators for determining the suitability of the product for such a purpose. This work reports on the effect of process conditions for oxidation of corn and waxy amaranth starch with film-forming ability as the major criterion. The process was followed using the analytical indicators of oxidation such as carboxyl content, chlorine consumption and ferricyanide number.