Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny

The prototypic chondrichthyan immunoglobulin (Ig) light chain type (type I) isolated from Heterodontus francisci (horned shark) has a clustered organization in which variable (V), joining (J), and constant (C) elements are in relatively close linkage (V-J-C). Using a polymerase chain reaction-based approach on a light chain peptide sequence from the holocephalan, Hydrolagus colliei (spotted ratfish), it was possible to isolate members of a second light chain gene family. A probe to this light chain (type II) detects homologs in two orders of elasmobranchs, Heterodontus, a galeomorph and Raja erinacea (little skate), a batoid, suggesting that this light chain type may be present throughout the cartilaginous fishes. In all cases, V, J, and C regions of the type II gene are arranged in closely linked clusters typical of all known Ig genes in cartilaginous fishes. All representatives of this type II gene family are joined in the germline. A third (kappa-like) light chain type from Heterodontus is described. These findings establish that a degree of light chain class complexity comparable to that of the mammals is present in the most phylogenetically distant extant jawed vertebrates and that the phenomenon of germline-joined (pre-rearranged) genes, described originally in the heavy chain genes of cartilaginous fishes, extends to light chain genes.     

Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco

Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons. To convert spectinomycin resistance from a noncell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance. Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5 untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds). When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished.     

The function of a ribosomal frameshifting signal from human immunodeficiency virus-1 in Escherichia coli

A 15-17 nucleotide sequence from the gag-pol ribosome frameshift site of HIV-1 directs analogous ribosomal frameshifting in Escherichia coli. Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. Limitation for phenylaianine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. Protein sequence analysis demonstrated the occurrence of two closeiy related frameshift mechanisms. In the first, ribosomes appear to bind leucyl-tRNA at the frameshift site and then slip leftward. This is the 'simultaneous slippage’mechanism. In the second, ribosomes appear to slip before binding amlnoacyl-tRNA, and then bind phenylaianyl-tRNA, which is encoded in the left-shifted reading frame. This mechanism is identicai to the‘overlapping reading’we have demonstrated at other bacterial frameshift sites. The HIV-1 sequence is prone to frame-shifting by both mechanisms in E. coli.     

The functional units of a peptostreptococcal protein L

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.     

Organosoluble enzyme-polymer complexes: A novel type of biocatalyst for nonaqueous media

Summary A novel type of organosoluble biocatalyst, representing a non-covalent complex of enzyme with a sugar-based amphiphilic polymer is described. The subtilisin Carlsbergpalmitoyl poly(sucrose acrylate) complex was found to be soluble and catalytically active in a number of organic solvents of different nature.     

Genetic diversity in and phylogenetic relationships of the Brazilian endemic cotton,Gossypium mustelinum (Malvaceae)

Gossypium mustelinum, one of five tetraploid species in the cotton genus, is geographically restricted to a few states in NE Brazil. Allozyme analysis was used to assess levels and patterns of genetic diversity inG. mustelinum and its relationship to the other tetraploid species. Genetic variation was low, with only 6 of 50 loci examined being polymorphic, a mean of 1.14 alleles per locus and a mean panmictic heterozygosity of 0.08. These estimates are low relative to other tetraploid cotton species, but are typical of island endemics. Interpopulational genetic identities were uniformly high, lending support to the concept of there being only one wild species of Brazilian cotton. The limited allelic diversity observed was correlated with geographical distribution, although variability is so limited in the species that geographically marginal populations are electrophoretically ordinary. Phylogenetic and phenetic analyses demonstrate thatG. mustelinum is isolated among polyploid cotton species, occupying one of the three basal clades resulting from an early radiation of polyploid taxa subsequent to polyploid formation. We suggest thatG. mustelinum represents a paleoendemic that presently exists as a series of widely scattered, relictual populations. Despite several centuries of sympatric cultivation ofG. barbadense andG. hirsutum, there was little evidence of interspecific introgression of alleles from cultivated cottons intoG. mustelinum.     

Overproduction of, and interaction within, bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.     

Spectroscopic studies of the manganese complex of Photosystem II

Our recent EPR and EXAFS experiments investigating the structure of the oxygen-evolving complex of PS II are discussed. PS II treatments which affect the cofactors calcium and chloride have been used to poise samples in modified forms of the S-states, S₁, S₂ and S₃. X-ray absorption studies indicate a similar overall structure for the manganese complex between treated and native samples although the influence of the treatments and cofactors is observed. Manganese oxidation (or oxidation of a ligand to the manganese cluster) is indicated to occur on each of the transitions S₁ S₂ and S₂ S₃ in these modified samples. The cluster appears to contain at least two inequivalent Mn-Mn pairs. In the native samples the Mn-Mn distance is 2.7 Å, but in samples where the calcium site is affected, one of the pairs has a 3.0 Å Mn-Mn distance. The intensity of the 3.3/3.6 Å interaction is reduced on sodium chloride treatment (calcium depletion) perhaps indicating calcium binding close to the manganese cluster. From EPR data we also propose that treatments which affect calcium and chloride binding cause a modification of the native S₂ state, slow the reduction of Y_z and allow an S3 EPR signal to be observed following illumination. The origin of the S3 EPR signal, a modified S₃ or S₂ X where X is an organic radical of unknown charge, is discussed in relation to the results from the EXAFS studies.Abbreviations EPR electron paramagnetic resonance spectroscopy - EXAFS extended X-ray absorption fine structure - HTG n-heptyl -d-thioglucoside - MES 2(N-morpholino)ethanesulfonic acid - OEC oxygen evolving complex - PPBQ phenyl-1,4-benzoquinone - PS II Photosystem II - Y_z redox active tyrosine     

Homologs of the yeast neck filament associated genes: isolation and sequence analysis of Candida albicans CDC3 and CDC10

Morphogenesis in the yeast Saccharomyes cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.     

Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS,of protein kinase C

Summary Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (I _K) and the M-current (I _M). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E₁ (PGE₁) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: I _k, a small current through a T-like Ca²⁺ channel, and no M-current.Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of I _k observed with cardiac cells, PKC activation produced only a small inhibition of I _k, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of I _k in neuroblastoma cells.Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.We thank Dr. Peter J. Parker for his generous gift of PKC, and Yvonne Vallis for her skillful assistance with the cultures and harvesting of the NG108-15 transfected cells.