Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Cardiovascular and renal effects of iso-rANP, a second natriuretic peptide from rat atria

Administration of a newly discovered second atrial peptide, iso-atrial natriuretic peptide (or iso-rANP(1-45) for the rat), caused hypotension, decreased heart rate, diuresis, and increased renal excretion of Na+, K+, and Cl- in the anesthetized rat. Bolus injections of chemically synthetic iso-rANP(1-45) had circulatory and diuretic activity equal to or greater than rANP(99-126) but, at low doses, a lesser effect on renal electrolyte excretion. The synthetic peptide fragment, iso-rANP(17-45), analogous in structure to rANP(99-126), had attenuated activity on the circulation, and at low doses, attenuated activity on the kidney. At higher doses, where renal responses to rANP(99-126) were less (downside of a biphasic response), both iso-rANP(1-45) and (17-45) had greater effects on water and electrolyte excretion than rANP(99-126). Injections of iso-rANP(1-45) and (17-45) increased hematocrit, whereas rANP(99-126) did not; furthermore, unlike rANP(99-126), iso-rANP did not affect arterial plasma Na+ concentration. The heart produces at least two genetically different atrial natriuretic peptides which affect the circulation and salt and water balance.     

Comparative seed ecology of the endangered shrub, Pimelea spicata and a threatening weed, Bridal Creeper: Smoke, heat and other fire-related germination cues

Summary Pimelea spicata R. Br. is a nationally listed endangered Australian shrub threatened with extinction by habitat fragmentation and environmental weed invasion. Bridal Creeper (Asparagus asparagoides L. W. Wight) is the primary weed threat to the largest remaining populations of P. spicata in the Cumberland Plain. Fire, as part of an integrated pest management program, offers the potential to stimulate P. spicata populations while controlling Bridal Creeper. It is important, therefore, to understand how the components of fire affect the germination and growth of both species. Using laboratory experiments we investigated the effects of smoke, heat, ash and/or light on the germination of P. spicata and Bridal Creeper. We found a significant promotive effect of smoke and indication of an inhibitory heat shock (90°C for 10 min) effect on the germination of P. spicata seeds. The response of Bridal Creeper seeds to the same factors was complex; while the results of one experiment suggested an inhibitory effect of smoke and a promotive effect of heat, subsequent trials were contradictory, implying that Bridal Creeper, like many weeds, is able to germinate under a wide range of environmental conditions. Other experiments investigated the optimal germination temperature and innate dormancy of P. spicata in the absence of fire‐related germination cues. Of the incubation temperatures investigated, the optimal diurnally fluctuating regime for P. spicata germinations was 10°C and 20°C in the night and day, respectively. The innate dormancy of freshly produced seeds disappeared after 3 months. In contrast to Bridal Creeper, we found a persistent germinable seed bank of about 97 P. spicata seeds/m² located in the top 5 cm of the soil profile. While fire alone is unlikely to kill Bridal Creeper plants, fire may help to manage local infestations of the weed by limiting germination and providing opportunity for herbicide treatment of regrowth.     

The pollen-specific LIM protein PLIM-1 from sunflower binds nucleic acids in vitro

The protein PLIM-1 (formerly SF3) from sunflower is expressed exclusively in mature, free pollen. It contains two LIM domains associated with an acidic C-terminus comprising six copies of the pentapeptide motif (A,T,S) (E,D) TQN. We have expressed the pollen protein as well as some of its mutant forms inEscherichia coli and have used the bacterially produced proteins to study interactions with nucleic acids. Our studies show that the protein binds DNA and RNA in vitro to form large complexes, while mutant polypeptides containing either a single LIM domain or a destabilized first or second LIM domain do not. Although these data suggest that the biological function of PLIM-1 involves interactions with nucleic acids, its role in pollen development remains unclear.     

Characterization of cpsF and its product CMP-N-acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli

Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N -acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al ., 1992). In this paper, we report the identification and characterization of cpsF , a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthetase encoded by the Escherichia coli K1 gene, neuA . The enzymatic function of the protein encoded by cpsF was established by cloning the gene in E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when a CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF , K1 antigen expression was restored. We conclude that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.     

Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue

The Serratia marcescens haemophore HasA is secreted by an ABC exporter comprising three envelope proteins. The ABC protein (ATP-binding cassette) HasD and the MFP protein (membrane fusion protein) HasE but not the outer membrane component have been isolated previously. In Escherichia coli , TolC, the outer membrane component of the haemolysin transporter, can form a hybrid exporter with HasD and HasE. This hybrid secretes HasA and the very similar metalloproteases from S. marcescens and Erwinia chrysanthemi . By analogy, the genuine exporter was predicted to secrete metalloproteases. The hasF gene was thus cloned from S. marcescens into an E. coli tolC mutant carrying hasD and hasE genes, by screening for a proteolytic phenotype on skimmed-milk plates. hasF encodes a protein sharing 74% identity with the E. coli TolC protein. Anti-TolC antibodies cross-reacted with a protein with an apparent molecular weight of 53 kDa in E. coli expressing hasF and in S. marcescens . hasF is unlinked to the has cluster and, unlike the has operon, is not iron regulated. hasF complements some of the tolC phenotypes, including drug- and detergent sensitivities and haemolysin secretion but not colicin E1 uptake. This suggests that the various functions of TolC could correspond to distinct domains on the protein.     

The control of respiration in wheat (Triticum aestivum L.) leaves

The aim of this work was to discover whether the respiration of wheat (Triticum aestivum L. cv. Huntsman) leaves, transferred to darkness after 7 h photosynthesis, showed an initial period of wasteful respiration. For young and old leaves, CO₂ production and O₂ uptake after 7 h photosynthesis were up to 56% higher than at the end of an 8-h night. The maximum catalytic activities of citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) at the end of the day did not differ from those at the end of the night. Changes in the contents of glucose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and -ketoglutarate did not as a group parallel the changes in the rate of respiration. The detailed distribution of label from [U-¹⁴C] sucrose supplied to leaves in the dark was similar at the end of the day and the end of the night. No correlation was observed between the rates of leaf respiration and extension growth. It is argued that the higher rate of respiration at the beginning of the night cannot be attributed to wasteful respiration.Abbreviation RQ respiratory quotient We thank Dr H. Thomas and Professor C.J. Pollock, Institute for Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, UK for their generous help in measuring leaf extension. R.H.A. thanks the Science and Engineering Research Council for a studentship.     

Identification and characterization of T-cell antigen receptor-related genes in phylogenetically diverse vertebrate species

Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene super-family in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems.The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers U22666 (Gd186cDNA), U22667 (Gd187cDNA), U22668 (Gd186), U22669 (Gd187), U22670 (Hf2A), U22671 (Hf191Y), U22672 (Hf191YcDNA), U22673 (Hf2AcDNA), U22674 (SnYYC191), U22675 (SnYYC193), U22678 (SnYYC193cDNA), U22679 (Xl11), and U23067 (SnYFC191)     

GROWTH OF HETEROSIGMA CARTERAE (RAPHIDOPHYCEAE) ON NITRATE AND AMMONIUM AT THREE PHOTON FLUX DENSITIES: EVIDENCE FOR N STRESS IN NITRATE-GROWING CELLS

The marine alga Heterosigma carterae Hulburt (Raphidophyta) was grown in N-limiting batch cultures using either nitrate or ammonium as the N source, at photon flux densities (PFDs) of 50, 200, and 350 μmol·m^-2·s^-1 in a 12:12 h LD cycle. Carbon content could be estimated from biovolume (μg C = 0.278 × nL; R = 0.98) but not reliably from pigment content. During exponential growth, ammonium-grown cells (in comparison with nitrate-grown cells at the same PFD) attained higher growth rates by at least 20%, contained more N, and had a lower C:N ratio, higher concentrations of intracellular free amino acids, and higher ratios of glutamine: glutamate (Gln: Glu) and asparagine: aspartate (Asn:Asp). Growth was nearly light-saturated on ammonium at 200 μmol·m^-2·s^-1 (cell-specific growth rate of 1.2 d^-1) but probably not saturated in nitrate-grown cells at 350 μmol·m^-2·s^-1. PFD did not affect Gln: Glu or Asn: Asp for a given N source. These results indicate that the nitrate-growing cells were more N-stressed than those using ammonium (which in contrast were relatively C-stressed) and that this organism would show an enhanced competitive advantage against other species when supplied with a transient supply of ammonium rather than nitrate .