• Volume 26, Issue 5

December 2001,   pages  547-698

• Clipboard: New challenges in human genetics: modifier genes

• Commentary: World views and Trojan horses in the sociobiology debate

• Commentary: The enigma of morphine tolerance: recent insights

• Where does our behaviour come from?

• Lens regeneration in mice under the influence of vitamin A

The effect of vitamin A has been studied on lens regeneration in young (7 days old) as well as adult mice. A longitudinal slit was made under local anesthesia in the cornea over the lens. The lens was extracted intact through the incision. Intraperitonial injection of vitamin A (0.05 ml of 30 IU/ml in young and 0.05 ml of 50 IU/ml in adult) was given to the operated animals. Vitamin A was found to induce lens regeneration in not only young but also in adult mice. Regenerated lenses were similar in shape, size, transparency and histological features to normal intact lenses.

• Homeotic regeneration of eye in amphibian tadpoles and its enhancement by vitamin A

After removal of both the lateral eyes of external gill stage tadpoles of the toad Bufo melanostictus, the pineal organ gets transformed into a median eye. This type of transformation occurrs in tadpoles of both control and vitamin A treated groups. However, vitamin A increases the likelihood of homeotic regeneration (57% in the control group and 71% in the vitamin A treated group). Histological studies showed that the newly transformed median eye developed from the pineal organ. The pineal eye so developed possessed all components of a normal eye such as a retina, sensory cells and lens.

• Local repeat sequence organization of an intergenic spacer in the chloroplast genome of Chlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: a complex mode of “copy-choice replication”?

Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA of Chlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt+) in a cross between a pair of polymorphic interfertile strains of Chlamydomonas (C. reinhardtii and C. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, P2” (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3′ end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, P2” seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely sequence-scrambled” product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a unique” new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a complex path” of copy-choice replication.

• MutS recognition: Multiple mismatches and sequence context effects

Escherichia coli MutS is a versatile repair protein that specifically recognizes not only various types of mismatches but also single stranded loops of up to 4 nucleotides in length. Specific binding, followed by the next step of tracking the DNA helix that locates hemi-methylated sites, is regulated by the conformational state of the protein as a function of ATP binding/hydrolysis. Here, we study how various molecular determinants of a heteroduplex regulate mismatch recognition by MutS, the critical first step of mismatch repair. Using classical DNase I footprinting assays, we demonstrate that the hierarchy of MutS binding to various types of mismatches is identical whether the mismatches are present singly or in multiples. Moreover, this unique hierarchy is indifferent both to the differential level of DNA helical flexibility and to the unpaired status of the mismatched bases in a heteroduplex. Surprisingly, multiple mismatches exhibit reduced affinity of binding to MutS, compared to that of a similar single mismatch. Such a reduction in the affinity might be due to sequence context effects, which we established more directly by studying two identical single mismatches in an altered sequence background. A mismatch, upon simply being flipped at the same location, elicits changes in MutS specific contacts, thereby underscoring the importance of sequence context in modulating MutS binding to mismatches.

• Role of LLD, a new locus for leaflet/pinna morphogenesis in Pisum sativum

Properties of a mutant at the LLD (LEAF-LET DEVELOPMENT) locus in pea Pisum sativum L. are reported in this paper. Plants homozygous for the Mendelian recessive mutation lld bear leaves in which a few to many leaflets are incompletely developed. Opposite pinnae of rachis nodes often formed fused incompletely developed leaflets. The lld mutation was observed to abort pinna development at almost all morphogenetic stages. The lld mutation demonstrated high penetrance and low expressivity. The phenotypes of lld plants in tl, tac, tl tac, tl af and tl af tac backgrounds suggested that LLD function is involved in the separation of lateral adjacent blastozones differentiated on primary, secondary and tertiary rachides and lamina development in leaflets. The aborted development of tendrils and leaflets in lld mutants was related to deficiency in vascular tissue growth. The morphological and anatomical features of the leaflets formed on a tl lld double mutant permitted a model of basipetal leaflet development. The key steps of leaflet morphogenesis include origin of the lamina by splitting of a radially symmetrical growing pinna having abaxial outer surface, opposite to the vascular cylinder, through an invaginational groove, differentiation of adaxial surface along the outer boundary of split tissue in the groove and expansion of the lamina ridges so formed into lamina spans.

• Gas chromatographic and mass spectrometric analysis of conjugated steroids in urine

This study was carried out qualitatively and quantitatively to investigate the presence and the concentrations of anabolic steroids in urine collected from orally administered humans. Microanalysis of conjugated steroids by gas chromatography and mass spectrometry (GC/MS) has been carried out. Following oral administration three major metabolites of anabolic steroid drugs have been detected and partially characterized. The six steroids can be analysed at the same time in 17 min. The lower detection limit was 10 ng/ml in 5 ml of urine. The conjugated steroids from urine were centrifuged to 2,430 𝑔 for 10 min, the supernatant solution passed through Amberlite XAD-2 column and the steroids eluted fraction esterified by using MSTFA and TMSI. The rate of metabolism and urinary excretion seem to be reasonably fast.

• Recombinant pre-pro-Concanavalin A (jack bean) is stable but of low solubility

The cDNA for pre-pro-Concanavalin A (pre-pro-ConA) was cloned into the cytoplasmic expression vector pKK233-2 to give rise to pCONEXP2 which was used to express the lectin precursor. Pre-pro-ConA is stable and is not transposed and ligated to form the mature protein. No signal peptide removal is observed. The solubility of pre-pro-ConA could not be increased by guanidine hydrochloride denaturation/dilution treatment.

• Randomly amplified polymorphic DNA-polymerase chain reaction analysis of two different populations of cultured Korean catfish Silurus asotus

Genetic similarity and diversity of cultured catfish Silurus asotus populations collected from two areas in western Korea were examined using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Out of 20 random primers tested, 5 produced 1344 RAPD bands ranging from 8.2 to 13.6 polymorphic bands per primer. The polymorphic bands in these populations ranged from 56.4% to 59.6%. Polymorphic bands per lane within populations ranged from 4.9% to 5.3%. The similarity within the Kunsan population varied from 0.39 to 0.82 with a mean (± SD) of 0.56 ± 0.08. The level of bandsharing values was 0.59 ± 0.07 within the catfish population from Yesan. The genetic similarity in cultured catfish populations may have been caused because individuals from two populations were reared in the same environmental conditions or by inbreeding during several generations. However, in view of bandsharing values, polymorphic bands and also the specific major bands that were inter-population-specific, significant genetic differentiation between these populations were present even if bandsharing (BS) values were somewhat numerically different. Therefore, the number of RAPD polymorphisms identified in this study may be sufficient to permit estimating genetic similarity and diversity. However, in future, additional populations, sampling sites and individuals will be necessary to make up for these weak points.

• Modelling studies on neurodegenerative disease-causing triplet repeat sequences d(GGC/GCC)n and d(CAG/CTG)n

Model building and molecular mechanics studies have been carried out to examine the potential structures for d(GGC/GCC)5 and d(CAG/CTG)5 that might relate to their biological function and association with triplet repeat expansion diseases. Model building studies suggested that hairpin and quadruplex structures could be formed with these repeat sequences. Molecular mechanics studies have demonstrated that the hairpin and hairpin dimer structures of triplet repeat sequences formed by looping out of the two strands are as favourable as the corresponding B-DNA type hetero duplex structures. Further, at high salt condition, Greek key type quadruplex structures are energetically comparable with hairpin dimer and B-DNA type duplex structures. All tetrads in the quadruplex structures are well stacked and provide favourable stacking energy values. Interestingly, in the energy minimized hairpin dimer and Greek key type quadruplex structures, all the bases even in the non-G tetrads are cyclically hydrogen bonded, even though the A, C and T-tetrads were not hydrogen bonded in the starting structures.

• Stress-induced evolution and the biosafety of genetically modified microorganisms released into the environment

This article is focused on the problems of reduction of the risk associated with the deliberate release of genetically modified microorganisms (GMMs) into the environment. Special attention is given to overview the most probable physiological and genetic processes which could be induced in the released GMMs by adverse environmental conditions, namely:

1. activation of quorum sensing and the functions associated with it,
2. entering into a state of general resistance,
4. stimulation of inter-species gene transfer.

To reduce the risks associated with GMMs, the inactivation of their key genes responsible for stress-stimulated increase of viability and evolvability is proposed.

• Subject Index

• Author Index

• Acknowledgements

• # Journal of Biosciences

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