Ligand requirements for Ca2+ binding to EGF-like domains.

Site-specific mutagenesis studies of the first epidermal growth factor-like (EGF-like) domain of human clotting factor IX suggest that the calcium-binding site present in this domain (dissociation constant Kd = 1.8 mM at pH 7.5 and ionic strength I = 0.15) involved the carboxylate residues Asp47, Asp49 and Asp64. To further characterize the ligands required for calcium binding to EGF-like domains, two new mutations, Asp47----Asn and Asp49----Asn, were introduced into the domain by peptide synthesis. 1H-NMR spectroscopy was used to obtain the dissociation constants for calcium binding to these mutations. Calcium binding to the Asp49----Asn modified domain is only mildly affected (Kd = 6 mM, I = 0.15), whereas binding to the Asp47----Asn modified domain is severely reduced (Kd = 42 mM, I = 0.15). From these data, it is proposed that the anionic oxygen atoms of the side chains of residues 47 and 64 are essential for calcium binding, whereas the side chain ligand for calcium at residue 49 can be a carboxyamide oxygen. As a control, the introduction of the modification Glu78----Asp in a region of the domain not believed to be involved in calcium binding had very little effect on the Kd for calcium (Kd = 2.6 mM, I = 0.15). Finally, the effect of an Asp47----Gly substitution found in the natural haemophilia B mutant, factor IXAlabama, was investigated. This peptide has a markedly reduced affinity for calcium (Kd = 37 mM, I = 0.15), suggesting that the defect in factor IXAlabama is due to impaired calcium binding to its first EGF-like domain.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

Influence of soil type on the transfer of plasmid RP4p from Pseudomonas fluorescens to introduced recipient and to indigenous bacteria

Abstract Transfer of plasmid RP4p from introduced Pseudomonas fluorescens to a co-introduced recipient strain or to members of the indigenous bacterial population was studied in four different soils of varying texture planted with wheat. Donor and recipient strains showed good survival in the four soils throughout the experiment. The numbers of transconjugants found in donor and recipient experiments in two soils, Ede loamy sand and Löss silt loam were significantly higher in the rhizosphere than in corresponding bulk soil. In the remaining two soils, Montrond and Flevo silt loam, transconjugant numbers were not significantly higher in the rhizosphere than in the bulk soil.
The combined utilization of a specific bacteriophage eliminate the donor strain and the pat sequence as a specific marker to detect RP4p was found to be very efficient in detecting indigenous transconjugants under various environmental conditions. The numbers of indigenous transconjugants were consistently higher in rhizosphere than bull soil. A significant rhizosphere effect on transconjugant numbers of transconjugants were recovered from Flevo and Montrond silt loam; these soils possess characteristics such as clay or organic matter contents which may be favorable to conjugation.     

The population dynamics of pike,Esox lucius,and perch,Perca fluviatilis,in a simple predator-prey system

Synopsis The population dynamics and predator-prey relationship of pike, Esox lucius, and perch, Perca fluviatilis, were examined in simple fish communities in two adjacent shallow lakes, Lochs Kinord and Davan, Deeside, Scotland. Few perch survive to age 3 but Z is low for fish > 3 years and perch live up to 17 years. Population fecundity remained relatively high and constant in perch because of the multi-age spawning stock and the presence of older more fecund perch. Growth rates of perch in both lochs are relatively high as a consequence of low stock abundance. The N, B, and P of adult perch were unusually low. The age range of pike, and N, B, P, and growth were in the range of values reported elsewhere. There was little variation in the strength of pike year classes and the importance of cannibalism and low occurrence of alternative prey in the lochs suggest that the populations were self-regulating. Cannibalism by adults was responsible for most of mortality in perch larvae, and predation by pike and adult perch was responsible for the majority of juvenile losses. This conclusion is supported by the high biomass ratios of pike:juvenile perch of 1.0–30.8. While the number of adult fish was almost equal, the biomass of adult pike was 2–3 × that of perch in Kinord and 6 × in Davan. In L. Kinord, where year class strength was stable, high predation pressure from perch and pike reduced perch abundance rather than eliminated year classes. Perch year classes fluctuated in abundance in L. Davan and were eliminated in the first summer in two sampling years. The pike, and particularly the perch populations, have features characteristic of fish communities in unperturbed ecosystems: namely, a wide range of age classes, stability in biomass with variation dampened by longevity, and low production.     

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I gene

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.     

Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2

Summary The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequencing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 protein sequence was found to be related to a family of proteins including hevein and Win-1, which have an amino-terminal lectin domain and a carboxy-terminal domain of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses characterized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dramatically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins.     

Characteristics of action potentials in Helianthus annuus

The action potentials induced by nondamaging electrical stimuli in 16- to 22-day-old plants of Helianthus annuus were examined. Typical recordings are presented. Mean values of their amplitudes and conduction velocities in the stem, the strength-duration relation, the 'all-or-none' law and the refractory periods have been determined. The amplitude and velocity of propagation were essentially identical in the upward and downward direction, but greater in the upper than in the lower half. In 'electrically active' plants, the rheobase value is 2 V, the minimum period for stimulation is 1.8 s. and the chronaxie 2.3 s. It is noted that the excitability level between similar plants on the same day and in the same plant on different days is highly variable and undergoes periodic changes.     

Cloning, sequencing and distribution of the Salmonella typhimurlum LT2 siaiidase gene, nanH, provides evidence for interspecies gene transfer

The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli. Sialidase expression was regulated positively by cAMP. In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity. A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S. typhimurium LT2. The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi. Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S. typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S. typhimurium obtained its nanH copy most recently from Salmonella arizonae. S. typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH. These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.