Bothrops jararaca snakes produce several bothrojaracin isoforms following an individual pattern

More than one isoform of bothrojaracin (BJC), a potent and specific thrombin inhibitor isolated from Bothrops jararaca venom, has been found in individual venoms collected from adult snakes. Variations in snake venom composition have previously been associated with factors such as age, sex, geographic origin, season of the year and diet. In order to obtain further information concerning individual patterns of expression of BJC isoforms, we have analyzed five individual Bothrops jararaca snake venoms collected at the same time from adult female snakes from the same geographic region. As expected, crude venoms showed a similar migration pattern on SDS-PAGE. BJC was purified using a procedure which includes an affinity chromatography step (PPACK-thrombin Sepharose). A slight variation in the amount of BJC obtained from individual venom samples was noticed. Inhibition of thrombin-induced platelet aggregation as well as migration pattern on SDS-PAGE (under reducing and non-reducing conditions) and isoelectric focusing varied considerably among BJC samples from the five snakes. The amino-terminal sequences (residues 1–34) of individual BJC samples were compared with the sequence deduced from isolated cDNAs encoding α and β chains of BJC. A high degree of homology was detected, although some residues differed from one sample to other. Altogether, data confirmed the heterogeneity found for BJC purified from individual snakes. Thus, the results indicate that: (1) individual specimens of Bothrops jararaca have different patterns of BJC isoform expression; and (2) it seems that genetic factors, at least in part, determine the variability found in BJC production.     

Transport properties of cuticular waxes of Fagus sylvatica L. and Picea abies (L.) Karst.: Estimation of size selectivity and tortuosity from diffusion coefficients of aliphatic molecules

Diffusion coefficients for thirteen lipophilic organic compounds in reconstituted waxes of Fagus sylvatica L. and Picea abies (L.) Karst. were measured to characterise the transport properties of the cuticular waxes that form the transport-limiting barrier of plant cuticles. Desorption kinetics (relative amounts desorbed versus time) were asymptotic, but could be linearized up to 50% desorption by plotting relative amounts desorbed versus the square root of time. Diffusion coefficients calculated from the slopes of the linear regressions ranged from 10^–22 to 10^–17 m²·s^–1 and decreased with increasing molecular size. This size dependency of diffusion coefficients was analysed, assuming an exponential dependence of the diffusion coefficients on molar volumes, which allowed cuticular transport properties to be related to the physical structure of the wax. Furthermore, the fact that the barrier properties of Fagus wax are less pronounced than those of Picea is interpreted as an ecological adaptation of the respective tree species to their habitats at the level of their cuticular transport barriers.Abbreviation D diffusion coefficient The authors gratefully acknowledge financial support by the Schwerpunkt Programm Baumphysiologie of the Deutsche Forschungsgemeinschaft.     

Satellite DNA and evolution of sex chromosomes

The satellite DNA (satellite III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (=1.700 g cm^–3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.     

Behaviour of sex chromosome associated satellite DNAs in somatic and germ cells in snakes

Sex chromosome associated satellite DNAs isolated from the snakes Elaphe radiata (sat III) (Singh et al., 1976) and Bungarus fasciatus (Elapidae) (minor satellite) are evolutionarily conserved throughout the suborder Ophidia. An autosome limited satellite DNA (B. fasciatus major satellite) is not similarly conserved. Both types of satellites have been studied by in situ hybridisation in various somatic tissues and germ cells where it has been observed that the W sex chromosome remains condensed in interphase nuclei. In growing oocytes however, the W chromosome satellite rich heterochromatin decondenses completely whilst the autosomal satellite rich regions remain condensed. Later, the cycle is reversed and the W chromosome condenses whilst the autosomal satellite regions decondense. In a primitive snake (Eryx johni johni) where the sex chromosomes are not differentiated and where there is no satellite DNA specific to them, these phenomena are absent. — The differential behaviour of autosomal and sex chromosome associated satellite DNAs is discussed in the light of gene regulation.     

Multiplication of the endangered Indian pitcher plant (Nepenthes khasiana) through enhanced axillary branching in vitro

Axenic seedling-derived two- to three-node stem segments of Nepenthes khasiana Hook.f. were successfully cultured on Woody Plant Medium containing 2.2 M benzyladenine to produce a 0.5–1.5 cm axillary shoot from each node in 7–8 weeks. The rapid growth along with the axillary branching of this shoot enabled amassing of 6–12 shoots during subculture. Excised shoots transferred to basal medium or rooted in medium containing 2.7 M naphthaleneacetic acid produced typical pitchers at leaf tips. Rooted plants were established in pots at 90–95% survival rate.Abbreviations AA ascorbic acid - AC activated charcoal - CA citric acid - BA 6-benzyladenine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - KC Knudson-C (1946) basal medium - MS Murashige & Skoog (1962) basal medium - WPM Woody Plant basal medium (Lloyd & McCown 1980)     

Bothrops jararaca fibrinogen and its resistance to hydrolysis evoked by snake venoms

Fibrinogen is an essential protein involved in several steps of hemostasis, being associated with the final steps of the blood coagulation mechanism. Herein, we describe the purification and characterization of a reptile fibrinogen, obtained from Bothrops jararaca plasma. Native B. jararaca fibrinogen showed a molecular mass of 372 kDa, and the reduced and alkylated fibrinogen molecule showed three chains of 71, 60 and 55 kDa, which are similar to the molecular masses of human and bovine Aα, Bβ and γ fibrinogen chains. Remarkably, B. jararaca fibrinogen was clotted by bovine thrombin, but B. jararaca, Crotalus durissus terrificus and Lachesis muta rhombeata venoms could not induce its clotting or hydrolysis. Thus, despite the similarities between B. jararaca and mammalian fibrinogens, the former shows distinctive features, which protect B. jararaca snakes from accidental envenomation.     

Effect of ammonium or nitrate on nitrogen fixation by a terrestrial Blue-green alga

Summary The effects of ammonium or nitrate-nitrogen on biological nitrogen fixation by an algal crust are compared. Nitrate-nitrogen up to 3.0 moles N g^–1 sand/algal crust at 60% water holding capacity did not affect fixation, whereas an ammonium-nitrogen concentration of 0.2 moles N g^–1 crust markedly depressed fixation. Consequences of these differential effects are considered.     

Behaviour of the ZW sex bivalent in the snake Bothrops jararaca

The behavior of the ZW sex bivalent was investigated in female meiosis of the poisonous snake Bothrops jararaca. The Z is euchromatic and synapses end to end with the W. The W chromosome shows a heterochromatic segment distally in the short arm. Pairing occurs between the long arm of the W and the slightly longer arm of the mediocentric Z. A sex vesicle, similar to the one found in the XY placental mammals, does not occur in snakes. The Z and W chromosomes segregate reductionally in the first meiotic division and equationally in the second.This work is dedicated to the memory of my father Lino Pires de Camargo     

Sex chromosome associated satellite DNA: Evolution and conservation

Satellites visible in female but not in male DNA were isolated from the snakesElaphe radiata (satellite IV, p = 1.708 g · cm^–3) andBungarus fasciatus (BK¹ minor, p=1.709 g · cm^–3). The satellites cross hybridize. Hybridization of³H labelled nick translated BK minor satellite DNA with the total male and female DNA and/or chromosomes in situ of different species of snakes revealed that its sequences are conserved throughout the snake group and are mainly concentrated on the W chromosome. Snakes lacking sex chromosomes do possess related sequences but there is no sex difference and visible related satellites are absent. The following conclusions have been reached on the basis of these results. 1. The W chromosome associated satellite DNA is related to similar sequences scattered in the genome. 2. The origin and increment in the number of the W satellite DNA sequence on the W chromosome is associated with the heterochromatinization of the W. 3. Satellite sequences have become distributed along the length of the W and resulted in morphological differentiation of sex chromosomes. 4. Evolutionary conservation of W satellite DNA strongly suggests that functional constraints may have limited sequence divergence.     

Ventilation and gas exchange in the diamondback water snake,Natrix rhombifera

Summary Simultaneous measurements of pulmonary and cutaneous oxygen and carbon dioxide exchange, pulmonary ventilation and heart rate were made on the diamondback water snake,Natrix rhombifera at 28°C using body plethysmography. Resting lung volume, maximum lung volume and tracheal volume were also measured.The following mean values were measured in undisturbed snakes breathing room air: total (pulmonary and cutaneous) O₂ uptake 46 mol · (kg min)^–1; total CO₂ output, 49 mol · (kg min)^–1; tidal volume, 12 ml (BTPS) · kg^–1; ventilatory rate, 6.9 min^–1; heart rate, 42 min^–1. From the measurements of tracheal volume, the effective (alveolar) ventilation was estimated as approximately 70% of total ventilation resulting in effective pulmonary and of 130 Torr and 20 Torr respectively. Cutaneous exchange accounted for 8.1% of the total and 12.4% of the total .Resting lung volume of anaesthetized snakes was 75 ml (BTPS) · kg^–1, maximum lung volume was 341 ml (BTPS) · kg^–1 and tracheal volume was 3.9 ml (BTPS) · kg^–1.     

Two type II keratin genes are localized on human chromosome 12

Summary Human genomic DNA containing two type II keratin genes, one coding for keratin 1 (K1, a 68-kD basic protein) and another closely linked type II gene 10–15 kb upstream (K?, gene product unknown), was isolated on a single cosmid clone. EcoRI restriction fragments of the cosmid were subcloned into pGEM-3Z, and specific probes comprising the C-terminal coding and 3 noncoding regions of the two genes were constructed. The type II keratin genes were localized by in situ hybridization of the subcloned probes to normal human lymphocyte chromosomes. In a total of 70 chromosome spreads hybridized with the K? probe (gHK?-3, PstI, 800 bp), 36 of the 105 grains observed were on chromosome 12, and 32 of these were clustered on the long arm near the centromere (12q11–13). In 100 labeled metaphases hybridized with the K1 probe (gHK1–3, BamHI-PstI, 2100 bp), 53 grains localized to chromosome 12 and 46 of these were found in the same region (q11–13). Therefore, both the gene for human keratin 1, a specific marker for terminal differentiation in mammalian epidermis, and another closely linked unknown type II keratin gene (K?, 10–15 kb upstream of K1) are on the long arm (q11–13) of human chromosome 12.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Complex interspecific hybridization in barley (Hordeum vulgare L.) and the possible occurrence of apomixis

Summary Several complex hybrids were produced from the combination [(Hordeum lechleri, 6x xH. procerum, 6 x) × H. vulgare, 2 x]. Crosses with six diploid barley lines resulted in triple hybrids, most of which had a full complement of barley chromosomes (no. 1–7), but were mixoploid with respect to alien chromosomes (19–22). In one combination, chromosome no. 7 was duplicated. Meiosis in triple hybrids showed low, but variable pairing (1.3–5.5 chiasmata per cell). The syndesis probably did not include the barley chromosomes. Direct back-crosses to di- and tetraploid barley lines were unsuccessful. Chromosome doubling of the triple hybrid based on cv Pallas resulted in a plant with 2n = 53–56, which had an increased fertility. Backcrosses to one di- and one tetraploid barley line resulted in offspring. The cross made with the tetraploid line (Haisa II), produced a 28-chromosomic plant in which the male parental genome was absent. We suspect that this plant may have arisen through parthenogenetic development of a reduced female gamete. The other cross with a diploid line (9208/9) resulted in plant with 2n = 51–53. The most likely explanation for this second plant is that an unreduced gamete from the amphiploid was fertilized by a normal gamete from the backcross parent, and during early embryo development, some chromosomes were eliminated.     

Bacterial genetic factors controlling the suppression of T4 phage amber mutants. II. Suppression patterns among the segregants of bacterial crosses

Summary Recombinant bacteria issuing from crosses between Hfr and F ^– E. coli strains which differ in their amber and non-amber-suppressor sensitive phage mutant suppression patterns exhibit the two parental phage suppression patterns and five other patterns. Analysis of the suppression patterns and comparisons of the chromosomal marker frequencies among the seven different recombinant classes permit identification of five distinct chromosomal regions which are sites of suppressor genes for which the parental strains carry different alleles: 1. the str region of the Hfr chromosome, 2. the ()-gal region of the F ^– chromosome, 3. the met-xyl region of the F ^– chromosome, 4. the thr region of the Hfr chromosome, and 5. the his-try region of the Hfr chromosome.The suppressor in the str region is probably coincident with the gene(s) determining the str phenotype of the parental Hfr strains. The suppressor residing in the ()-gal region of the F ^– chromosome appears to be the su _II glutamine-inserting suppressor. The quantitative expression of su _II appears to be reduced by the presence of the Str^r mutation carried by the F ^– parent, and this reduced efficiency of suppression can be counteracted progressively by the presence of the suppressor residing in the met-xyl region of the F ^– chromosome and of the two suppressors residing in the thr and his-try regions of the Hfr chromosome.     

Close karyological kinship between the reptilian suborder serpentes and the class aves

Summary In contrast to the situation found in two classes of warm-blooded vertebrates, mammals and birds, the class Reptilia is not uniform with regard to total genetic content; rather, it contains two distinct categories. The close cytological kinship between snakes and birds was revealed. Both are almost identical in total genetic content, which is about 50 per cent that of placental mammals. Both have microchromosomes, as well as Z-chromosomes very similar in absolute size, comprising nearly 10 per cent of the homogametic haploid (AZ) set. This leads to the implication that snakes and birds originated from the same lineage, and that their Z-chromosomes have not changed substantially since the Jurassic period of the Mesozoic era, about 180 million years ago.Within the reptilian suborder Serpentes, the step-by-step differentiation from the primitive ZW pair to the grossly heteromorphic ZW pair could be observed. In the ancient family Boidae, the sex chromosomes were still homomorphic to each other. In the family Colubridae, the beginning of heteromorphism was manifested in two ways. In some species, a pericentric inversion on the W caused it to differ from the Z; in others, duplication of the W occurred. In the family Crotalidae, the W had apparently achieved its very specialized status; it was a distinctly smaller element.In Säo Paulo, this work was supported by Fundacão de Amparo a Pesquisa do Estado de São Paulo e Fundo de Pesquisas do Instituto Butantan. In Duarte, this work was supported in part by grant CA-05138-05, National Cancer Institute, U. S. Public Health Service. Contribution No. 36-64, Department of Biology, City of Hope Medical Center.     

Interspecific hybrids between Romanian and Kurdistan hamsters

From crossings between Mesocricetus brandti and Mesocricetus newtoni 23 hybrids were obtained which presented characters intermediate between the parents and which were completely sterile. The number of chromosomes in the hybrid animals (2n=40) is also intermediate between the parents and the karyotype presents two distinct chromosome sets corresponding to those in the two parent species. The regular presence of multivalents at diakinesis and metaphase I probably causes the non balanced disjunction of the genetic material, and thus, probably, the total sterility of the hybrids. The study of sex chromatin showed the existence of a sex dimorphism, the female presenting more nuclei with 1–2 sex chromatin bodies. The presence of 2 sex chromatin bodies of different sixes may be explained by the heteroehromatinization of a whole X chromosome and half of its homologue.Histological study showed that the male and female genital apparatus are intensively affected in hybrids and the spermatogenetic and oogenetic activity is generally stopped. This determines the total sterility of the hybrids.     

Preferential generalized transduction by bacteriophage Mu

Summary The generalized transduction by bacteriophage Mu was found to be preferential for the 0–1 min segment of the E. coli K12 chromosome. This transduction pattern is obtained with phage lysates grown on all F^-, F⁺ and Hfr tested, and is not marker-specific.Phages grown by both lytic infection and by heat induction of prophages at different locations of the host's chromosome show the same transduction pattern, indicating that generation of transducing DNA does not directly depend on excision events. Conjugation of independently obtained Muc ⁺-lysogenic strains of HfrC with a multiauxotrophic F^- recipient strain lysogenic for a Mucts62 prophage, shows that transfer of the temperature-resistance character (Muc ⁺) is not preferentially linked to the 0–1 min segment. The lysogenizing integrations do therefore not take place within the segment preferentially transduced by the phage.A model¹ for the generation of the transducing DNA is proposed, which assumes that for its replication, Mu DNA is integrated close to the 0–1 min segment of the host chromosome, which is then preferentially replicated and packaged into the phage heads.     

Karyotype evolution and sex chromosome differentiation in schistosomes (Trematoda,Schistosomatidae)

The morphology of C-banded metaphase chromosomes has been studied in two hermaphroditic and ten gonochoristic digenetic trematodes (schistosomes). Comparison of numbers and morphology of chromosomes indicates that the karyotype of primitive trematodes probably was composed of 10 (or 11) pairs of telocentric or subtelocentrie chromosomes, and reduction of chromosome numbers in advanced species resulted from centromeric fusion rather than elimination of chromosomes. Observation of heteromorphic chromosomes in a hermaphroditic trematode (Spirorchis) suggested a differentiation of pre-sex chromosomes in species ancestral to dioecious trematodes which possess distinctly differentiated sex chromosomes. Our results indicate that differentiation of Z and W chromosomes in the gonochoristic trematodes resulted from: (a) partial constitutive heterochromatinization of the W chromosome (Schistosoma mansoni and S. haematobium complexes, African schistosomes), (b) deletion of part of the W (S. japonicum and S. mekongi, Asian schistosomes), and (c) translocation of part of one sex chromosome onto another (Schistosomatium douthitti and Heterobilharzia americana, American schistosomes) with subsequent heterochromatinization of the W in H. americana.     

Minichromosomes inDrosophila melanogaster derived from the transposing elementTE1

A minichromosome has originated from the transposing elementTE1. This autonomously replicating chromosome contains the structural genes white and roughest, from theDrosophila X chromosome. It arose within a stock carryingTE1 at 45F on chromosome2. In addition to thew andrst genes, the minichromosome may carry section 45C–45F from chromosome2. It is inherited by 33%–47% of the offspring. By this criterion it carries a centromere, although the origin of the centromere is unknown. From this minichromosome a still smaller one has originated, probably through the loss of all material from chromosome2 together with some heterochromatin. At the same time a duplication of white and roughest could have taken place. This chromosome has a strange morphology and is more frequently lost in meiosis than the larger one, but is still transmitted to about 29%–37% of the progeny of one parent heterozygous for the minichromosome. In both cases the flies have variegated eyes, probably as a result of position-effect variegation. The variegation pattern is influenced by factors in theX chromosome. The size of the smaller minichromosome is little more than 1 Mb as determined by pulsed field gel electrophoresis.     

Linkage of Genes Controlling Morphological Traits with Isozyme Markers of Rye Chromosomes

Data on linkage of 12 rye genes controlling morphological traits (el, Vs, ln, w, np, ct2, Hs, Ddw, cb, mn, vi1, mp) with one or several isozyme markers of individual rye chromosomes (2R–7R) are presented. Linkage of the following gene pairs was established: chromosome 2R: Est3/5–el, el–-Glu, Sod2–el, Sod2–Vs; chromosome 3R: ln–Got4; chromosome 4R: w–Got1, np–Got1; chromosome 5R: Est4–ct2, Est6/9–ct2, ct2–Est2, ct2–Aco2, Est2–Hs, Aco2–Hs, Est2–Ddw, Aco2–Ddw; chromosome 6R:Lap2–cb, cb–Aco1, Est10–mn; chromosome 7R: Acph2/3–vi1, Got2–vi1, mp–Acph2/3. The reasons for mapping a very small number of genes in rye in spite of high intraspecific variability of this species are discussed. An approach is suggested to improve this situation by simultaneous identification and mapping of all diverse spontaneous mutations maintained in heterozygous state in various rye cultivars.