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.

Globin mRNA in Friend cells: its structure, function and synthesis.

Accumulation of haemoglobin in induced MEL cells begins with the activation of transcription of the globin genes. Though much has been learned of cellular events affecting expression of the globin genes, e.g., from noninducible variants of MEL cells and cell fusion between MEL cells and other cell types, there is at present no in vitro system available that would permit more detailed study of the molecular events leading to transcription of the globin genes. Presumably, with the availability of cloned chromosomal genes such systems will soon be found. The products of transcription detected in induced MEL cells are 15 S and 11 S species which are precursor forms of beta- and alpha-globin mRNA, respectively. An unmodified primary transcript has not been detected. The 15 S species possesses a fully methylated 'cap' 1 structure the 5' end and poly(A) at the 3' end. Conceivably, there could be cleaving or splicing events preceding the 'capping' and polyadenylation, but all these reactions must occur extremely rapidly, since with a t 1/2 approx. 2 min a large proportion of the 15 S beta-globin RNA must be newly synthesised. It also contains the two intervening sequences found in the chromosomal genes. Selective processing occurs since from pulse and pulse-chase experiments most if not all of the 15 S beta-globin RNA is processed to mature 10 S beta-globin RNA very rapidly, whereas less than 10% of newly synthesised nuclear RNA (HnRNA) leaves the nucleus, the remainder being hydrolysed in the nucleus with a t 1/2 approx. 20 min. Perhaps rapid processing permits efficient transport to the cytoplasm. Further processing occurs in steps; apparently the large intervening sequence is removed first followed by the small intervening sequence. These steps do not appear to be rate limiting events and these sequences have not been detected separately from the 15 S beta-globin RNA. Such results and the wide divergency of intervening sequences, suggest that the intervening sequences per se play no essential function in the cell, though their presence in the nuclear transcript appears to necessary for processing to the mRNA. The selective processing accounts for more increase of the globin RNA in MEL cells, and further accumulation occurs by virtue of the stability of globin mRNA (t 1/2 approx. 17 h) compared with the bulk of poly(A)-RNA (t 1/2 approx. 3 h). It would appear, however, that specific destabilization of a class of stable mRNA (t 1/2 approx. 35 h) is necessary to allow globin mRNA to asccount for 90% of the mRNA population in reticulocytes.

Nucleotide sequence of mouse 5-aminolevulinic acid synthase cDNA and expression of its gene in hepatic and erythroid tissues.

The cDNA coding for 5-aminolevulinic acid (ALA) synthase (EC 2.3.1.37) in both liver and anemic spleen of the mouse has been cloned. The liver clone was selected by complementation of an Escherichia coli hemA mutant. Erythroid clones were obtained by screening a cDNA library made from mouse anemic spleen RNA, using the liver cDNA as a probe. The sequences of the spleen-derived and liver-derived cDNAs are identical. The nucleotide sequence and predicted amino acid (aa) sequence of a 1.85-kb spleen-derived cDNA is presented. The mouse ALA synthase as sequence displays extensive homology to ALA synthase of chick embryonic liver. The ALA synthase mRNA, detected by Northern blot analysis, was the same size, approx. 2.3 kb, in mouse liver, anemic spleen, and mouse erythroleukemia cells. It is therefore unlikely that different isozymic forms of ALA synthase are present in mouse erythroid and hepatic tissue and this is not the basis for the different effects of heme and porphyrinogenic compounds on the expression of liver and erythroid ALA synthase.

The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II.

The nucleotide sequence of a clone containing mouse carbonic anhydrase (CA) cDNA in pBR322 has been determined. The cloned cDNA contains all of the coding region except for nucleotides specifying the first eight amino acids, and all of the 3' noncoding region, which consists of 700 nucleotides. A cDNA clone was identified which contains an additional 54 bp at the 5' end, so that the complete amino acid sequence of mouse CA could be deduced. This sequence showed a 73-81% homology with other mammalian CA form II isozymes, 56-63% with form I isozymes, and 52-56% with form III isozymes. By examination of the amino acids which are unique and invariant for each isozyme, the mouse amino acid sequence was found to contain 16 of the 23 residues that are unique and invariant to mammalian CA form II isozymes, but only one or no residue for forms I and III, respectively.

Sequence comparison of human and murine erythrocyte alpha-spectrin cDNA.

The results of hybridization analyses using cDNA probes for mouse and human alpha-spectrin mRNA indicate that a single gene encodes the alpha-subunit of erythrocyte spectrin. Sequencing of the cDNA clones showed that they code for 370 amino acids (aa) covering three repeat domains close to the C terminus of alpha-spectrin. The cloned cDNAs will now permit the isolation of the alpha-spectrin gene and should lead to the characterization of the genetic aspects in human hereditary anemias in which alpha-spectrin has been characterized as the site of the molecular defect.

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.

Cloning of rabies virus matrix protein mRNA and determination of its amino acid sequence.

A cDNA clone of mRNA for rabies virus matrix (M) protein has been identified. The clone hybridizes to an mRNA species from rabies virus-infected cells, whose size correlates to the size of the M protein in rabies virions, and selects an mRNA that translates into a polypeptide corresponding in size to M protein. The nucleotide sequence of the cloned cDNA was determined and from this a complete amino acid sequence for M protein was deduced. The deduced sequence of 202 amino acids bears no detectable sequence homology with vesicular stomatitis virus M protein although these proteins may share functional homology.

Plasma levels of secretin in man and dogs: validation of a secretin radioimmunoassay.

We have developed and validated a secretin radioimmunoassay that is sufficiently sensitvie to measure circulating levels of secretin in the plasma of man and dogs. At a final dilution of 1:50,000, the antibody bound 30 percent to 40 percent of radioiodinated (125 I) 6-tyrosyl synthetic secretin. Pure natural porcine secretin was used as a reference standard and a linear dose-response curve was generated with 10 to 1,000 pg. of the polypeptide. Little or no cross-reactivity was found when graded doses of other gastrointestinal polypeptides were assayed in the radioimmunoassay and immunoreactive secretin (IRS) in volumes of serum up to 300 mul could be measured accurately.

Catabolism of secretin by the liver and kidney.

We have investigated the roles of the liver and the kidney in the catabolism of secretin, using a specific and sensitive radioimmunoassay. Dogs were prepared with sampling catheters in the aorta, hepatic vein, portal vein, and renal vein and with electromagnetic flow probes on the portal vein, hepatic artery, and renal artery. Secretin levels in the vessels entering and leaving the liver and kidney were determined by radioimmunoassay and the total mass of secretin [concentration (picograms per milliliter) X plasma flow rate (milliliter per minute)] was calculated during an intravenous infusion of exogenous secretin and during release of endogenous secretin by acidification of the proximal intestine. The total masses of secretin entering and leaving the liver were the same during secretin infusion and during the release of endogenous secretin. Under conditions of elevation of plasma secretin, however, the kidney extracted 30 percent of arterial secretin during secretin infusion and 45 percent during release of endogenous secretin. Clearly the kidney is a major site of secretin catabolism.

The effect of nephrectomy on the catabolism of secretin.

Previous studies have shown the kidney to be an important site for the catabolism of secretin. We have investigated possible sites within the kidney for secretin uptake in four intact anesthetized dogs. The disappearance half-time of an intravenous infusion of secretin, measured by a sensitive and specific ratioimmunoassay in four intact anesthetized dogs was 2.84 minutes. After ureteral ligation (to arrest glomerular filtration), the half-time of a similar intravenous dose of secretin was 2.78 minutes. Finally, the renal vascular pedicles were ligated to totally exclude the kidneys from the circulation and the half-time was found to be 5.43 minutes. These findings demonstrate that the efficient renal mechanisms for secretin degradation are not dependent upon glomerular filtration but upon some other mechanism, presumably located in renal tubular cells.

Amino acid sequence of the rabies virus glycoprotein deduced from its cloned gene.

Double-stranded complementary DNA was synthesized from rabies virus-specific glycoprotein mRNA and inserted into the Pst I site of pBR322. The glycoprotein inserted sequence contains approximately 1.75 kilobase pairs and lacks only approximately 35 nucleotides from the 5' terminus of the glycoprotein mRNA. The nucleotide sequence indicates a polypeptide of 524 amino acids beginning with an initiation codon ATG and ending with a termination of codon TGA. The first 19 amino acids make up a signal peptide preceding the sequence lys-phe-pro-ile-tyr-thr- which has been identified by the N-terminal analysis of amino acids of the purified rabies virus glycoprotein.

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.

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.

Cloning of mouse carbonic anhydrase mRNA and its induction in mouse erythroleukemic cells.

Mouse carbonic anhydrase mRNA was detected in poly(A+) RNA of anemic spleens sedimenting as a RNA species at 14 S. Subsequently, poly(A+) RNA (12-16 S) was used as a template for the synthesis of double-stranded cDNA, which was inserted into the PstI site of pBR322 by oligo-dG:dC tailing. A recombinant plasmid containing carbonic anhydrase cDNA was identified by a positive hybridization selection assay and by partial DNA sequencing. Predicted amino acid sequences showed homology with the known sequences of rabbit and human carbonic anhydrase I and II. The clone contained sequences for most of the coding region and 600-700 base pairs at the 3' noncoding region of the mRNA. Hybridization analysis of poly(A+) RNA from uninduced and induced mouse erythroleukemic cells labeled for short and long time periods indicated that induction results in an increase of carbonic anhydrase mRNA in newly synthesized RNA.

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.