Glacier recession alters stream water quality characteristics facilitating bloom formation in the benthic diatom Didymosphenia geminata.

Glaciers provide cold, turbid runoff to many mountain streams in the late summer and buffer against years with low snowfall. The input of glacial meltwater to streams maintains unique habitats and support a diversity of stream flora and fauna. In western Canada, glaciers are anticipated to retreat by 60-80% by the end of the century, and this retreat will invoke widespread changes in mountain ecosystems. We used a space-for-time substitution along a gradient of glacierization in western Canada to develop insights into changes that may occur in glaciated regions over the coming decades. Here we report on observed changes in physical (temperature, turbidity), and chemical (dissolved and total nutrients) characteristics of mountain streams and the associated shifts in their diatom communities during de-glacierization. Shifts in habitat characteristics across gradients include changes in nutrient concentrations, light penetration, temperatures, and flow, all of which have led to distinct changes in diatom community composition. Importantly, glacial-fed rivers were 3-5 °C cooler than rivers without glacial contributions. Declines in glacial meltwater contribution to streams resulted in shifts in the timing of nutrient fluxes and lower concentrations of total phosphorus (TP), soluble reactive phosphorus (SRP), and higher dissolved inorganic nitrogen (DIN) and light penetration. The above set of conditions were linked to the overgrowth of the benthic diatom Didymosphenia geminata. These changes in stream condition and D. geminata colony development primarily occurred in streams with marginal (2-5%) to no glacier cover. Our data support a hypothesis that climate-induced changes in river hydrochemistry and physical condition lead to a phenological mismatch that favors D. geminata bloom development.

Selection of genetic inhibitors of rabies virus.

A cDNA library of short random fragments derived from four of the five genes of the rabies virus genome has been used to isolate genetic suppressor elements (GSEs) expressed intracellularly that inhibit rabies virus replication. Two nucleotide fragments, one from the rabies virus nucleocapsid protein (N) gene and the other from the phosphoprotein (P) gene, have been identified as inhibitors of rabies virus replication in cell culture. The N cDNA fragment is expressed in sense-orientation and could produce a dominant negative protein affecting virus replication. The P cDNA fragment is expressed in the inhibitory antisense direction. Inhibition of rabies virus replication was detected in cell culture using an ELISA for detection of rabies virus glycoprotein expression on the cell surface and immunofluorescence for detection of intracellular rabies virus N expression. Both the sense and antisense GSEs, because of their targeted inhibition of rabies virus replication, have possible uses in rational design of antiviral compounds for treatment of rabies. This approach could be applied to any virus, particularly to those that lyse their target host cell.

Effects of natural organic matter source on reducing metal toxicity to rainbow trout (Oncorhynchus mykiss) and on metal binding to their gills.

Juvenile rainbow trout (Oncorhynchus mykiss, 3 g) were exposed for 74 h in ion-poor (soft) water to a mixed-metal solution in the presence of 4, 6, and 10 mg C/L natural organic matter (NOM). The metals were 0.2 microM Pb, 0.1 microM Hg, 0.1 microM Cd, 1.3 microM Cu, 0.05 microM Ag, and 3.5 microM Co, and the natural organic matter was isolated by reverse osmosis from three sources in southern Ontario, Canada. The six-metal solution alone was extremely toxic to the fish. Increasing concentrations of each NOM increased trout survival, but the NOM having the most allochthonous properties (from Luther Marsh) increased fish survival most, while the NOM having the most autochthonous properties (from Sanctuary Pond, Point Pelee) increased fish survival least. This pattern was reflected in the degree of reduction of Pb and Cu accumulation by the gills. Relatively simple chemical characterization of NOM, such as protein-to-carbohydrate ratios, or optical characterization, such as absorbance-to-fluorescence ratios (e.g., representing aromaticity), may adequately reflect these biologically relevant differences in organic matter quality.

Physical properties of a single-motif erythrocyte spectrin peptide: a highly stable independently folding unit.

Spectrin is a long flexible rod-like actin cross-linking protein mostly comprised of many tandem homologous 106-residue motifs. In this study, the conformational stability and physical properties of a single homologous motif peptide, alpha1, were evaluated and compared to intact spectrin monomers and alphabeta heterodimers. It is interesting that while spectrin dimers elongate by about 3-fold in low ionic strength buffers relative to their size in physiological buffers, the single-motif peptide does not show significant changes in secondary structure in 10 mM phosphate buffer compared with isotonic buffer. This single-motif peptide is monomeric in physiological buffer as demonstrated by equilibrium sedimentation studies, and its hydrodynamic radius determined by gel filtration and dynamic light scattering of about 2.2 nm is consistent with an elongated rod-like shape. Unfolding of the single-motif peptide in urea solutions was similar to unfolding of intact heterodimers. Differential scanning calorimetry analyses showed that this single motif undergoes a reversible two-state transition with a Tm of 53 degrees C and an enthalpy of 65 kcal/mol in physiological buffer. Thermal stability was unaffected by ionic strength changes, but was decreased below physiological pH. These data show that this 13 kDa spectrin motif is a monomeric, highly stable, triple-helical, independently folding protein building block with physical characteristics that define many of the structural properties of the 526 kDa spectrin heterodimer. In contrast, interactions between adjacent motifs are probably responsible for spectrin's molecular flexibility and elasticity.

Molecular basis and haplotyping of the alphaII domain polymorphisms of spectrin: application to the study of hereditary elliptocytosis and pyropoikilocytosis.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are inherited disorders of erythrocyte shape that are frequently associated with abnormalities in alpha-spectrin, one of the principal structural proteins of the erythrocyte membrane skeleton. Five polymorphisms of the alpha-spectrin gene, located in a 6-kb interval of genomic DNA, were identified and analyzed in normal and mutant alpha-spectrin alleles. Three of these polymorphisms are due to single nucleotide substitutions in the alpha-spectrin gene coding region that lead to changes in the amino acid sequence. In combination, these three polymorphisms are responsible for the different peptide phenotypes of the alphaII domain previously observed following limited tryptic digestion of spectrin protein. The most common haplotype, type 1, was found predominantly in Caucasians and was the only haplotype identified in Asians. Haplotypes 2, 3, and 4 were identified predominantly in individuals of African ancestry and were commonly found in patients with HE or HPP. Analysis of coinheritance of alphaII domain polymorphisms with alpha-spectrin gene mutations causing HE or HPP in African-American patients with HE and HPP suggests that, with one exception, a given HE/HPP mutation is present in an alpha-spectrin gene of only one haplotype, indicating a founder effect. The other two polymorphisms located in this region of the alpha-spectrin gene do not change the amino acid sequence of the encoded alpha-spectrin chain and are not in linkage disequilibrium with three of the four alphaII domain haplotypes. A model is proposed for the evolutionary origin of the different haplotypes.

Mapping the human erythrocyte beta-spectrin dimer initiation site using recombinant peptides and correlation of its phasing with the alpha-actinin dimer site.

Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of beta-spectrin with a complementary region near the C-terminal of alpha-spectrin (Speicher, D. W., Weglarz, L., and DeSilva, T. M. (1992) J. Biol. Chem. 267, 14775-14782). Both spectrin subunits consist primarily of tandem, 106-residue long, homologous, triple-helical motifs. In this study, the minimal region of beta-spectrin required for association with alpha-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent beta-spectrin peptides was particularly critical. The beginning of the first homologous motif for both beta-spectrin and the related dimerization site of alpha-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif beta-spectrin peptide (beta1-4+) with this earlier starting point bound to full-length alpha-spectrin with a Kd of about 10 nM, while deletion of these first 8 residues reduced binding nearly 10-fold. N- and C-terminal truncations of one or more motifs from beta1-4+ showed that the first motif was essential for dimerization since its deletion abolished binding, but beta1+ alone could not associate with alpha-monomers. The first two motifs (beta1 2+) represented the minimum lateral dimer assembly site with a Kd of about 230 nM for interaction with full-length alpha-spectrin or an alpha-spectrin nucleation site recombinant peptide, alpha18-21. Each additional motif increased the dimerization affinity by approximately 5-fold. In addition to this strong inter-subunit dimer association, interactions between the helices of a single triple-helical motif are frequently strong enough to maintain a noncovalent complex after internal protease cleavage similar to the interactions thought to be involved in tetramer formation. Analysis of hydrodynamic radii of recombinant peptides containing differing numbers of motifs showed that a single motif had a Stokes radius of 2.35 nM, while each additional motif added only 0.85 nM to the Stokes radius. This is the first direct demonstration that spectrin's flexibility arises from regions between each triple helical motif rather than from within the segment itself and suggests that current models of inter-motif connections may need to be revised.

The exon-intron organization of the human erythroid beta-spectrin gene.

The human erythrocyte beta-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid beta-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the beta-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present beta-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat beta 10 is encoded by 4 exons. No single position of an intron in the beta-spectrin gene is conserved between any of the 17 beta-spectrin and 22 alpha-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5' donor splice-site sequence begins with GC. The beta-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid beta-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp.

Functional characterization of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site.

Spectrin, a heterodimer composed of alpha and beta subunits, interacts with itself head-to-head to form tetramers in the erythrocyte membrane cytoskeleton. The NH2-terminal region of alpha-spectrin, encompassing the alpha I 80-kDa domain, was expressed in Escherichia coli. In addition to the correctly initiated polypeptide, four smaller polypeptides were produced by initiation at internal codons. Only the full-length polypeptide was able to bind to spectrin dimers, beta monomers, and to a recombinant polypeptide containing the COOH terminus of beta-spectrin. The head-to-head interaction with beta-spectrin was also retained by a recombinant polypeptide containing the NH2-terminal 158 amino acids of the alpha subunit. Deletion of the first 27 or 49 NH2-terminal amino acids abolished binding of this polypeptide to the beta monomer. The phasing used to design these recombinant polypeptides was based on a conformational model recently refined by Speicher et al. (Speicher, D. W., DeSilva, T. M., Speicher, K. D., Ursitti, J. A., Hembach, P., and Weglarz, L. (1993) J. Biol. Chem. 268, 4227-4235), where the structural unit begins and terminates around residue 30 of the repeat unit. The binding properties, mobility on gel filtration, and circular dichroism data of the recombinant polypeptides indicated that most polypeptides were able to assume their native conformation.

Purification and characterization of a nuclear DNA-binding factor complex containing topoisomerase II and chromosome scaffold protein 2.

In a search for factors that influence the process of erythroid differentiation at the molecular level, we have identified UB2, a nuclear protein factor that was originally observed for its ability to bind to a very specific and highly conserved sequence motif present in human, mouse, rabbit, and chicken beta-globin genes, as well as carbonic anhydrase I, c-myb, and the immunoglobulin heavy chain enhancer region. It was also observed for its appearance in undifferentiated but not differentiated mouse erythroleukemia cells. Purification of UB2 by DEAE-cellulose chromatography and repeated passages through a DNA affinity column, revealed a complex pattern with three major components of 170, 116, and 48 kDa, respectively. The 170-kDa protein was identified as topoisomerase (topo) II by Western blot analysis, catalytic assays, and antibody interference with UB2 binding. The complex topo II in UB2, however, has a more stringent sequence requirement for DNA binding than does topo II. The 116-kDa protein has been determined to be a proteolytic product of topo II. The chromosome scaffold protein 2 (135 kDa) copurified with UB2, and anti-scaffold protein 2 serum inhibited UB2 binding to DNA.

Production of a functional monoclonal antibody recognizing human colorectal carcinoma cells from a baculovirus expression system.

The light and heavy chain cDNA of a murine monoclonal antibody (MoAb) with specificity for human colorectal carcinoma cells have been expressed separately, together, and as a dual construct in insect cells infected with recombinant baculoviruses. High levels of the MoAb were expressed under the control of the polyhedrin promoter. The antibody maintained its specific binding to human colorectal carcinoma cells and mediated lysis of these cells by human lymphocytes, monocytes, and murine macrophages, as determined in antibody-directed cellular cytotoxicity (ADCC) assays. The recombinant immunoglobulin (Ig), like its ascitic counterpart, did not mediate lysis by either human or rabbit complement. The expression of a recombinant antibody exhibiting both functional binding site and Fc region capacities shows that the baculovirus system could be employed in the production of therapeutic Ig.

Evaluation of foreign gene codon optimization in yeast: expression of a mouse IG kappa chain.

We have optimized the codons in an immunoglobulin kappa chain gene to those preferred in the yeast Saccharomyces cerevisiae. The mutant and wild type kappa chain genes were each fused with a synthetic invertase signal peptide that also contained only yeast-preferred codons, and expressed in the F762 yeast strain. The use of yeast-preferred codons resulted in a more than 5-fold increase in the rate of synthesis and at least a 50-fold increase in the steady state level of protein.

A differentiation stage-specific factor interacts with mouse carbonic anhydrase form I gene and a conserved sequence in mammalian beta-globin genes.

We have identified in mouse erythroleukemic (MEL) cells a novel factor which binds to the 3' flanking region of beta-globin genes. Upon induction, this DNA binding factor disappears as beta-globin gene transcription increases. The factor protects 20-30 base pairs (bp) of a sequence which is tightly conserved in beta-globin genes including chicken, human, mouse and rabbit. A very similar sequence binds the factor in the mouse carbonic anhydrase form I gene, whose transcription diminishes upon induction of MEL cells. The factor, or a closely related form, is detected in nonerythroid cells and binding sites have been detected in other genes, including c-myb and immunoglobulin heavy chain-enhancer. We suggest that this factor could play a role in determining the timing of gene expression in several differentiating cell types.

The exon-intron organization of the human erythrocyte alpha-spectrin gene.

The human erythrocyte alpha-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlapping lambda recombinants. The exon-intron junctions were identified and the exons mapped. The gene is encoded by 52 exons whose sizes range from 684 bp to the smallest of 18 bp. The donor and acceptor splice site sequences match the splice site consensus sequences, with the exception of one splice site where a donor sequence begins with -GC. The size and location of exons do not correlate with the 106-amino-acid repeat, except in three locations where the surrounding codons are conserved as well. The lack of correspondence between exons and 106-amino-acid repeat is interpreted to reflect the appearance of a spectrin-like gene from a minigene early in the evolution of eukaryotes. Since current evidence indicates that introns were present in genes before the divergence of prokaryotes and eukaryotes, it is possible that the original distribution of introns within the minigene has been lost by the random deletion of introns from the spectrin gene.

Structure of a mouse erythroid 5-aminolevulinate synthase gene and mapping of erythroid-specific DNAse I hypersensitive sites.

The enzyme 5-aminolevulinate synthase (ALA-S) catalyzes the first step in heme biosynthesis. In this study, the mouse erythroid gene has been cloned and analyzed in order to investigate the regulation of ALA-S expression during erythroid differentiation. The gene spans approximately kbp and consists of 11 exons and 10 introns. The first exon is 37 bp, non-coding, and followed by a 6kb intron. The mRNA capsite was mapped by primer extension and defines a promoter that contains no apparent TATA element. S1 nuclease analysis detects the presence at low levels of a 45 bp-deleted form of the ALA-S mRNA created by the use of an alternative splice site at the intron 2/exon 3 junction. Five DNAse I hypersensitive sites were detected in chromatin from uninduced and induced MEL cells. One site is at the promoter; the others are in the body of the gene. No significant differences were observed in the patterns or intensity of the hypersensitive sites in the uninduced and induced MEL cells, however, no sites in ALA-S were observed in NIH 3T3 cells or in deproteinized DNA. Thus, these sites are specific for erythroid chromatin but appear to be established at an earlier stage of differentiation than represented by the uninduced MEL cell.

Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA.

A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library. A mouse beta-spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli. This clone was used to probe total RNA from various mouse tissues. Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb. Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA. All of these bands could be detected after hybridization under both stringent and nonstringent conditions. This suggests that erythroid beta-spectrin may also be expressed in the brain. No bands could be detected in kidney, liver, or spleen RNA. Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions. Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin. The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.

Specific pattern of gene expression during induction of mouse erythroleukemia cells.

We have studied the expression of several characterized genes during induction of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide (DMSO) and have observed a specific pattern of changes in transcriptional activity and steady-state RNA levels associated with erythroid differentiation. During induction there is a gradual, steady decrease in total transcriptional activity and RNA content per cell, which by day 3 of DMSO treatment amounts to less than 50% of the level in the uninduced cell. During this time we observe increases in transcriptional activity for 5-aminolevulinic acid synthase, carbonic anhydrase form II, and band 3 coordinate with the large increase in beta-globin gene transcription. The results also demonstrate an early decrease in transcription for carbonic anhydrase form I, which precedes decreases in transcription for glyceraldehyde phosphate dehydrogenase and rRNA genes. Changes in steady-state RNA levels reflected changes in transcriptional activity during induction except for carbonic anhydrase II mRNA. These results represent the first report characterizing the regulated expression at transcriptional and posttranscriptional levels of several known genes that are characteristically expressed in the erythrocyte. The results demonstrate that coordinate gene expression in erythroid differentiation occurs primarily at the level of transcription.

Assignment of mouse beta-spectrin gene to chromosome 12.

The structural gene for the beta-subunit of the mouse erythrocyte spectrin, hereinafter designated as Sp-b, was assigned to the mouse chromosome 12. This assignment was made by Southern analysis of genomic DNA from mouse X Chinese hamster hybrid cells using cloned mouse erythrocyte beta-spectrin cDNA as a probe. In the PstI-digested genomic hamster cell DNA a single band of 2.0 kb was detected, whereas PstI-digested mouse DNA gave a band of 4.2 kb, when probed with the mouse erythroid beta-spectrin cDNA clone. This allowed us to analyze a panel of mouse X Chinese hamster somatic cell hybrids to map this gene to chromosome 12. Interestingly, this assignment is different from that observed for the alpha-subunit of spectrin, which has been mapped to chromosome 1 in mouse. These results serve as a basis for further genetic characterization of the mouse hemolytic anemias.

Chromosomal localization of a human band 3-like gene to region 7q35----7q36.

Band 3, the major transmembrane protein of erythrocytes, mediates the exchange of anions across the membrane and anchors the erythroid membrane skeleton. Proteins immunologically related to Band 3 have been detected in a variety of nonerythroid cells. We have isolated a human cDNA clone that encodes a protein related to but distinct from the erythroid form of Band 3, based on the comparison of the amino acid sequence for the two proteins. The presence of the gene for the Band 3-like protein in a panel of mouse-human somatic cell hybrids containing subsets of human chromosomes correlated with the presence of human chromosome 7. In situ hybridization analysis using the c-DNA for this nonerythroid Band 3 gene further localized the gene to region 7q35----7q36 of human metaphase chromosomes.