Gerald Bergtrom

Clicker Sets as Learning Objects

Science courses were among the earliest adopters of student response systems, more commonly called clickers, because they engage students in collaborative activities on a scale not heretofore possible in large lecture halls. Standard explanatory slides are interspersed with interactive questions to which students enter responses by pressing a key on a response device (the clicker). By criteria found to be acceptable in several studies, a series of these standard and interactive slides that encourage critical thinking about important concepts or experiments constitutes a limited content learning object. Examples of paired, matched-content sets of non-interactive and clicker (i.e., interactive) slides for use by students at home and by the instructor in class are presented to illustrate uniquely powerful, mutually reinforcing learning objects.

Accumulation of cadmium by the fourth instar larva of the fly Chironomus thummi.

Accumulation and effects of cadmium were investigated in Chironomus thummi larvae exposed to 10, 100 and 250 mug radiolabeled Cd/1 for up to 4 days. (1) After 4 days, average cadmium accumulation was 6.6 ng Cd/mg dry weight (10 mug Cd/1 exposure) and 177 ng Cd/mg dry weight (250 mug Cd/1 exposure). (2) Dissection studies showed that by 32 hr of exposure to both cadmium concentrations, 63.5-81.4% of accumulated cadmium was confined to the posterior midgut epithelium. Light microscope autoradiography similarly showed accumulations of cadmium in posterior midgut epithelium and smaller amounts in fat body and muscle. Little cadmium was associated with Malphigian tubules, haemocoel, anterior midgut or exoskeleton. (3) After exposure to 10 or 250 mug Cd/1, 60-75% of cadmium in ultracentrifuged homogenates of whole animals or dissected guts was associated with the resulting supernatant. When supernatants were further analyzed by gel chromatography, cadmium eluted with both a high and low molecular weight peak. The relative proportions of cadmium in the two peaks varied with concentration and length of exposure. (4) Transmission electron microscopy of posterior midgut cells from animals exposed to cadmium demonstrated frequent mitochondrial lesions. Exposure to high cadmium concentrations caused some posterior midgut cells to undergo generalized structural degeneration.

Intron-containing globin genes in the insect Chironomus thummi

All previously reported chironomid globin genes are intronless, suggesting that the ancestral chironomid globin gene was also intronless. In this study, the coding regions of the closely linked Chironomus thummi globin (Gbs) IIβ and IX genes are shown to be interrupted by noncoding DNA bounded by a 5′-GT and a 3′-AG. Both genes have appropriately placed transcription and translation signals. Polymerase chain reactions on genomic DNA with oligonucleotides flanking and within the putative Gb IIβ intron generated products the size predicted for a gene with a 64-nucleotide intron, and sequencing of a cloned PCR fragment also revealed the intron. A partial-length Gb IIβ cDNA sequence exactly matches that of the Gb IIβ coding regions. We conclude that the intron-containing chironomid globin genes are functional. Regions of the Gb IIβ and IX genes spanning the introns are more similar (86%) than the exons themselves (72% similarity), possibly due to partial gene correction. Surprisingly, Gb II β and IX gene homologues in C. tentans are intronless. If the common ancestor of chironomid globin genes was not intronless, introns were lost in at least three C. thummi globin-gene lineages, and more recently by Gb II β and Gb IX genes in C. tentans. If, as previously believed, the ancestral chironomid globin gene was intronless, then an intron was recently acquired in only one C. thummi globin sublineage. These alternatives are discussed.

Structure of the larval midgut of the fly Chironomus thummi and its relationship to sites of cadmium sequestration.

The midgut structure of 4-7 day old fourth instar Chironomus thummi larvae was investigated with the light arid electron microscopes. Four regions are present which may be identified by the following major features: (1) Anterior I: this is the region under the esophageal invagination. (2) Anterior II: short microviili characterize these cells. Long narrow, basal plasma membrane infoldings associated with mitochondria are conspicuous. This region is hypothesized to be important in ion and fluid transport. (3) Anterior III: numerous crystals are seen in these cells. Storage is proposed to be a major function of this region. (4) Posterior: these cells have long microviili, extensive RER, many Golgi, and short basal plasma membrane infoldings. Posterior cells probably function in secretion of digestive enzymes and absorption of nutrients. Cadmium is sequestered by the midgut, almost exclusively in the posterior cells. This phenomenon is discussed.

Regulation of plasminogen activation by interleukin-6 in human lung fibroblasts

We determined that exposure of cultured lung fibroblasts (HEL-299) to recombinant human interleukin-6 (0-400 ng/ml) resulted in a dose- and time-dependent increase in secreted and cell lysate PAI-1 and total tPA levels (maximal increase of 2.6-fold and 1.7-fold, respectively). Specificity of this response was indicated when increases in PAI-1 levels were inhibited by neutralizing polyclonal antibodies to IL-6, but not with non-specific antibodies. Inhibition of the response to IL-6 by cycloheximide and alpha-amanitin indicates that increases in PAI-1 are dependent on both protein and RNA synthesis. The addition of IL-6 to HEL-299 cells also stimulated a dose- and time-dependent increase in steady-state PAI-1 mRNA levels (3.8 to 15.1 pg/micrograms total RNA by 24 h). A rapid increase (5-6-fold) in PAI-1 mRNA levels was found between 3 and 12 h. Nuclear run-on assays using a maximum dose of IL-6 showed that IL-6 increases a 4-fold rate of transcription of the PAI-1 gene. We further showed that LPS induces a 70% increase in secreted IL-6 and a 50% increase in PAI-1 protein levels. Increasing doses of anti-IL-6 completely blocked the effect of LPS on PAI-1 while non-specific antibodies had no effect. These studies suggest an autocrine role for IL-6 in regulating localized proteolysis and modulating tissue remodeling during acute inflammatory conditions by fibroblasts.

Multiple clustered genes of the haemoglobin VIIB subfamily of Chironomus thummi thummi (Diptera)

The nucleotide and inferred amino acid sequence of three globin VIIB genes from the midge, Chironomus thummi thummi have been determined. These three genes are intronless, like all previously reported Chironomid globin genes. Transcription of globin genes VIIB-4, VIIB-5 (and/or VIIB-6) and VIIB-7 was demonstrated by S1 nuclease protection analysis. Comparison of actual and inferred globin VIIB amino acid sequences reveals that (i) most of the amino acid substitutions are conservative in nature, and (ii) none of the substitutions are expected to interfere with the ability of the globins to bind haem. Nucleotide sequence comparisons suggest that gene duplications and partial gene corrections (conversions) have occurred recently in the globin VIIB subfamily locus.

Evolution of Orthologous Intronless and Intron-Bearing Globin Genes in Two Insect Species

Abstract. While globin genes ctt-2β and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2β and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species.

An electrospray ionization mass spectrometric study of the extracellular hemoglobins from Chironomus thummi thummi.

The aquatic larvae of the dipteran, Chironomus thummi thummi contain extracellular hemoglobins which exhibit stage-specific expression. We have used maximum entropy-based deconvolution of the complex, multiply charged electrospray ionization mass spectra, to demonstrate the presence of more than 20 components, ranging in mass from 14,417.3 Da to 17,356.5 Da in the 4th instar larvae. Of the 15 major peaks with intensities > 10 relative to 100 for the 14,417.3 Da-component (CTT-IV), only the 15,528.2-Da peak does not correspond to a known amino acid sequence. Since the number of C. thummi thummi globin genes now stands at 27, including one cDNA and not counting three that must encode known globins, our results suggest that only a limited number of the globin genes are expressed in the 4th instar larvae.

Differential regulation of insect globin and actin mRNAs during larval development in Chironomus thummi

S1 nuclease protection assays were used to measure changes in the steady-state levels of six different globin (Gb) mRNAs in the midge, Chironomus thummi thummi (C. thummi, Diptera) during larval development. Two distinct patterns of change were observed. GbI, IV, VIIB-4 and VIIB-5 transcripts were present in 3rd instar larvae, rose from low levels immediately post-moult to peak levels by day 2-3 of the 4th instar, and then declined, reaching near-basal levels by day 7-8. In contrast, transcripts of GbIII (known from previous studies to be 4th instar-specific) and VI, which were undetectable in the 3rd instar, rose to high levels by day 2 of the 4th instar, but remained elevated thereafter. Our data further showed that closely linked Gb genes were not necessarily expressed in a coordinate manner. Unlike the Gb mRNAs, actin (Act) mRNA levels (measured by slot-blot hybridization to a heterologous probe) increased progressively as a proportion of total RNA during 4th instar development. Therefore, the regulation of C. thummi Gb transcript levels is specific, differing from that of Act and among the Gb mRNAs themselves. Elevated 20-hydroxyecdysone (HE) titer at the 3rd-4th instar moult correlates with the low steady-state levels of Gb mRNAs immediately post-moult. However, other aspects of Gb mRNA profiles cannot be explained on the basis of a direct repressive effect by HE on Gb gene transcription.

Sequence and evolution of the gene for the monomeric globin I and its linkage to genes coding for dimeric globins in the insect Chironomus thummi

We isolated genomic clones containing sequences encoding globins I and IA from a Chironomus thummi thummi genomic library. Three clones contain globin IA (ctt-1A) genes, while one contains a globin I (ctt-1) gene. The coding regions of the four genes are identical except for the single base substitution accounting for the globin I/IA polymorphism. The noncoding DNA flanking the coding region is more than 98% similar, confirming a previous hypothesis that the globin ctt-1 and ctt-1A genes are alleles. Hemoglobins I and IA are monomeric in the insect hemolymph. Earlier in situ hybridization studies suggested that monomeric and dimeric globin genes are clustered at different chromosomal loci. In situ hybridization of ctt-1 DNA to polytene salivary gland chromosomes places the ctt-1 gene on the same band as genes for the dimeric globins IIβ and VIIB, forcing revision of the earlier hypothesis that genes for monomeric and dimeric globin genes are at different loci. The evolution of the ctt-1 and ctt-1A alleles and of the two globin gene loci are discussed.