Evolution of the Larval Cuticle Proteins Coded by the Secondary Sex Chromosome Pair: X2 and Neo-Y of Drosophila miranda: II. Comparison at the Amino Acid Sequence Level

The larval cuticle proteins (LCPs) are encoded by a multigene family, Lcp1–4, located at the right arm of the metacentric autosome 2 (2R) in Drosophila melanogaster. Due to a chromosome fusion the Lcp locus of Drosophila miranda is situated on a pair of secondary sex chromosomes, the X2 and neo-Y chromosomes. Comparing the deduced amino acid sequences of the autosomal D. melanogaster loci with the sex-chromosomal loci of D. miranda, we were able to trace the evolution of the Lcp loci with respect to their different chromosomal inheritance. The length of the signal peptide is conserved in all four LCPs, while the size of the mature LCPs varies. Conserved protein motifs became obvious from the alignment, indicating regions of structural and functional importance. Analyzing intra- and interspecific sequence similarities of the Lcp gene families allowed us to reconstruct the phylogeny of the gene cluster. Alignment with cuticle amino acid sequences originating from divergent insect species reveals motifs already present in the primordial insect LCPs. These motifs indicate different levels of constraint acting during the evolution of the LCPs. Received: 27 December 1995 / Accepted: 30 April 1996     

A Chromatin-associated Kinesin-related Protein Required for Normal Mitotic Chromosome Segregation in Drosophila

The tiovivo (tio) gene of Drosophila encodes a kinesin-related protein, KLP38B, that colocalizes with condensed chromatin during cell division. Wild-type function of the tio gene product KLP38B is required for normal chromosome segregation during mitosis. Mitotic cells in tio larval brains displayed circular mitotic figures, increased ploidy, and abnormal anaphase figures. KLP38B mRNA is maternally provided and expressed in cells about to undergo division. We propose that KLP38B, perhaps redundantly with other chromosome-associated microtubule motor proteins, contributes to interactions between chromosome arms and microtubules important for establishing bipolar attachment of chromosomes and assembly of stable bipolar spindles.     

Isolation and characterization of a novel mesophilic,fresh-water methanogen from river sediment Methanoculleus oldenburgensis sp. nov.

A new mesophilic, irregular coccoid methanogen isolated from a river sediment is described. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. For growth acetate is strictly required. Elevated levels of sodium chloride were not required and were inhibitory at concentrations above 1.5% (w/v). The optimal growth temperature was at 45°C. The DNA base ratio was 48.6±1 mol% G+C. The polar lipid pattern and the polyamine content were similar to that found in several Methanoculleus species. The new isolate CB-1 was assigned as Methanoculleus oldenburgensis (DSM 6216).     

Structure and Glycosylation Patterns of Surface Proteins from Woodchuck Hepatitis Virus

Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans.     

TWO CLASS I ALDOLASES IN KLEBSORMIDIUM FLACCIDUM (CHAROPHYCEAE): AN EVOLUTIONARY LINK FROM CHLOROPHYTES TO HIGHER PLANTS1

Two fructose-bisphosphate aldolases(EC 4.1.2.13) from Klebsormidium flaccidum Silver, Mattox and Black-well were purified by affinity elution from phosphocellulose. The two enzymes were subsequently separated by HPLC on an anion-exchange column (QAE-silica). The aldolase eluting first represented 5% of the total activity; the other aldolase represented the remaining activity. The activity of the enzymes was not reduced by the presence of 1 mM EDTA or increased by 0.1 mM Zn²⁺, establishing their character as class I type (Me²⁺ independent) aldolases. The K_m(fructose-1,6-bisphosphate) values were 1.7 and 34.7 μM for the enzyme eluting first and second, respectively, from the QAE-silica column. The subunit molecular masses, as determined by SDS-PACE, were 40.5 and 37 kD; the specific activities of the purified enzymes were 7.9 and 24.7 · mg^?1 protein, respectively. The two aldolases of K. flaccidum are homologous to the cytosol and chloroplast specific isoenzymes of higher plants by several criteria and are therefore probably located in the same cellular compartments in K. flaccidum. The K_m and specific activity for the chloroplast aldolase of K. flaccidum are three times higher than for the chloroplast aldolase of higher plants, a remarkable difference. Immunotitration with specific antisera against the chloroplast aldolase of Chlamydomonas reinhardtii Dangeard and spinach showed that the chloroplast aldolase of K. flaccidum was immunochemically intermediate in structure to the respective aldolases of C. reinhardtii and higher plants. K. flaccidum is the second species of Charophyceae (besides Chara foetida Braun) with two class I aldolases as in higher plants whereas two species of Chlorophyceae have only one class I aldolase and, under some conditions, an additional class II (Me²⁺ dependent) aldolase. Thus, aldolases may turn out, in addition to the known enzymes of glycolate conversion and urea degradation, be a novel enzyme system to evaluate algal evolution along with cytological features.     

Stable Production of Human Insulin-like Growth Factor 1 (IGF-1) in the Milk of Hemi- and Homozygous Transgenic Rabbits Over Several Generations

One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 ± 12g/ml (ranging from 223 ± 61 to 484 ± 39 g/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 ± 41 g/ml (ranging from 360 ± 15 to 678 ± 80 g/ml). Homozygous huIGF-1 females clearly showed a significantly increased production performance of the recombinant protein.     

Two novel mutations in exons 19a and 20 and a BsaI polymorphism in a newly characterized intron of the neurofibromatosis type 1 gene

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. A total of 170 unrelated NF1 patients were screened for mutations in four exons by temperature-gradient gel electrophoresis. Preparatory work revealed the presence of a previously uncharacterized intron (19a) in what was previously designated exon 19; this allowed us to develop assays for genomic mutation screening in the newly defined exons 19a and 19b. Two novel NF1 mutations were detected: a single-base insertion in exon 19a creating a frameshift, and a second mutation affecting the splice donor site of intron 20 and leading to skipping of exon 20. A novel BsaBI polymorphism was identified in intron 19a. Received: 11 August 1997 / Accepted: 13 November 1997     

The occurrence of Rhincalanus gigas,Calanoides acutus,and Calanus propinquus (Copepoda: Calanoida) in late May in the area of the Antarctic Peninsula

Summary The Antarctic copepod species Rhincalanus gigas, Calanoides acutus and Calanus propinquus were studied in the area of the Antarctic Peninsula in May 1986. Research was focussed on vertical distribution and stage composition of the populations. Rhincalanus gigas occurred in greatest densities in the upper layers of the water column, and copepodite stages CI and CII and nauplii dominated the population. Gut content analyses suggest that R. gigas was actively feeding. Copepodite stage CV dominated the Calanoides acutus population. At two deep basin stations (water depth>1000 m) the C. acutus population occurred below 500 m, whereas at shallower stations the majority was found above 300 m. Most specimens had empty guts. Calanus propinquus occurred in low densities, mainly in the upper water layers, and copepodite stage CV dominated. Most individuals of stage V had food in their guts. Our results suggest that C. acutus had ceased feeding and was overwintering in a resting stage (diapause), while C. propinquus and R. gigas were still active, the latter species having finished a late autumn spawning.