Induction of skin fibrosis in mice expressing a mutated fibrillin-1 gene

BACKGROUND: Tight skin mice (TSK) bear a mutated Fibrillin-1 (Fbn-1) gene. Genetic studies show that the TSK mutation is closely associated with the Fbn-1 locus (0-0.7 cM). A previous study showed two recombinants between the Fbn-1 locus and the TSK mutation. TSK mutation and mutated Fbn-1 gene cosegregate in F1 mice. MATERIALS AND METHODS: To elucidate the role of the mutated Fbn-1 gene in occurrence of TSK syndrome, we generated transgenic (Tg) mice expressing mutated Fbn-1 gene. In another set of experiments, we injected normal mice after birth with a plasmid bearing mutated Fbn-1 gene (pdFbn-1). RESULTS: Our results demonstrate that the pdFbn-1 Tg mice developed permanent cutaneous hyperplasia that was permanent. In mice injected as newborns with a plasmid bearing the sense pdFbn-1 gene, cutaneous hyperplasia was transient. In contrast to TSK mice, neither Tg nor mice injected with plasmid developed lung emphysema. The pdFbn-1 Tg and TSK mice spontaneously produced anti-topoisomerase I and anti-Fbn- antibodies, as do humans afflicted by scleroderma; whereas, those injected with a plasmid containing the pdFbn-1 gene produced only anti-Fbn-1 autoantibodies. CONCLUSIONS: The results suggest that, although cutaneous hyperplasia is due to mutated Fbn-1 gene, the TSK syndrome may be multifactorial.     

Intergenic transcripts containing a novel human cytochrome P450 2C exon 1 spliced to sequences from the CYP2C9 gene

The cytochrome P450 2C (CYP2C) gene locus was found to includea novel exon 1 sequence with high similarity to the canonicalexon 1 of CYP2C18. Rapid amplification of cDNA ends (RACE) andPCR amplifications of human liver cDNA revealed the presenceof several intergenic species containing the CYP2C18 exon 1–likesequence spliced to different combinations of exonic and intronicsequences from the CYP2C9 gene. One splice variant was foundto have an open reading frame starting at the canonical translationinitiation codon of the CYP2C18 exon 1–like sequence.Another variant consisted of the nine typical CYP2C9 exons splicedafter the CYP2C18 exon 1–like sequence through a segmentof CYP2C9 5' flanking sequences. Moreover, analysis of bacterialartificial chromosome (BAC) clones revealed that the CYP2C18exon 1–like sequence was located in the intergenic regionbetween the CYP2C19 and CYP2C9 genes. The finding that a solitaryexon is spliced with sequences from a neighboring gene may beinterpreted as representing a general evolutionary mechanismaimed at using the full expression potential of a cell's genomicinformational content.     

A Cold-Sensitive spo14 Mutation Affecting Ascospore Formation in the Fission Yeast Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, twenty sporulation-specificgenes (spo1–spo20) have been identified and analyzed.We found that a mutation designated spo14–221 caused cold-sensitivesporulation: ascospores were formed at 30?C but not at 23?C.Nuclear staining with 4',6-diamidino-2-phenylindole revealedthat a strain with this mutation completed meiosis even at therestrictive temperature. Electron microscopy showed that assemblyof forespore membranes during meiosis II was abnormal and incompletein the mutant cultured at 23?C. Temperature-shift experimentsindicated that the cold-sensitive period began during earlymeiosis I and terminated with the end of meiosis II. These resultssuggest that the product of the spo14 gene is synthesized andexecutes its function prior to the expression of the sporulation-deficientphenotype of the mutant, prior to the formation of the abnormalforespore membrane. (Received October 5, 1989; Accepted February 28, 1990)     

Genomic organization and expression of hamster UDP-N-acetylglucosarmine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase

The hamster gene for uridine diphosphate N-acetyl-D-glucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase(L-G1PT) was found to extend over 6.5 kb and to contain nineexons. The exons ranged in size from 63 to 214 bp, encodingthe 408 amino acid protein. The introns ranged from 85 bp to1.4 kb. Upstream 5' sequences included two possible TATA boxes,one possible CCAAT box and at least two potential GC boxes.Heterologous expression was successful in Schizosaccharomycespombe, and resulted in cells that were tunicamycin resistantand had 12-fold more L-G1PT activity than wild-type cells. Antiserumprepared to a hydrophilic peptide (residues 300–320) ofthe L-G1PT protein reacted with a 35–36 kDa protein inmembrane samples from Chinese hamster ovary (CHO) cells andS.pombe cells that had increased levels of L-G1PT activity.In both cases, antigenic peptide competed with the 35–36kDa protein detected by the antiserum. N-acetylglucosarmine 1-phosphate transferase dolichol glycosylation     

Analysis of the Promoter of the Auxin-Inducible Gene, parC, of Tobacco

The auxin-responsive region (AuxRR) in the promoter of the parCgene was analyzed in transgenic tobacco plants in which the5' flanking region of the parC promoter was placed upstreamof the gene for rß-glucuronidase (GUS). The AuxRRwas located between nucleotides (nt) –226 and –54.Detailed dissection of this segment revealed that the presenceof the non-contiguous sequences from nt –226 to –151and from nt –84 to –54 was required for the expressionof the auxin responsiveness of the parC promoter. The sequencefrom nt –226 to –151 was found to contain a sequencewhich resembles the as-1 element in the 35S promoter of cauliflowermosaic virus (CaMV). Although it has been reported that theas-1 element is involved in auxin responsiveness [Liu and Lam(1994) J. Biol. Chem. 269: 668], we showed that introductionof a point mutation into the as-1-like sequence completely eliminatedauxin responsiveness, a result that suggests that the sequenceis indispensable for auxin responsiveness. However, the presenceof the as-1-like sequence alone was not sufficient for auxinresponsiveness, since the segment (nt –226 to –84)that included the as-1-like sequence failed to confer auxinresponsiveness on the core promoter. It is possible that thetwo separately located sequences play specific roles in interactionswith trans-factors that are required for the expression of theauxin responsiveness of the parC promoter. (Received March 11, 1996; Accepted July 9, 1996)     

The Genetic Basis of Preference for Sweet Substances Among Inbred Strains of Mice: Preference Ratio Phenotypes and the Alleles of the Sac and dpa Loci

Five inbred strains (129/J, BALB/cByJ, C3HeB/FeJ, C57BI/6J andDBA/2J) were examined with two-bottle (48 h) preference ratiotesting across concentrations of sodium saccharin (3 x 10^–4M, 10^–3 M, 3 x 10^–3 M and 10^–2 M), d-phenylalanine(10^–3 M, 10^–2 M and 10^–1 M), and l-glutamine(10^–2 M, 3 x 10^–2 M, 10^–1 M and 3 x 10^–1M). Three consistent groupings of strains were observed acrosssubstances and concentrations:

1. C57BI/6J (preference at low andhigh concentrations);
2. BALB/cByJ and C3HeB/FeJ (preferenceat high concentrations);
3. 129/J and DBA/2J (preference at highconcentration for sodiumsaccharin and indifference to d-phenylalanineand l-glutamine).

If a single locus (presumably dpa or Sac) determines these phenotypes,there are likely to be three alleles. If two independent loci(presumably dpa and Sac) determine these phenotypes, an allelicassignment of Sac^b/dpa^+s for the C57BI/6J strain, Sac^b/dpa^–sfor the BALB/cByJ and C3HeB/FeJ strains, and either Sac^d/dpa^+sor Sac^d/dpa^–s for the 129/J and DBA/2J strains is suggested.Chem. Senses 20: 291–298, 1995.     

A global assessment of mesozooplankton respiration in the ocean

Published data on the biomass and specific respiration ratesof mesozooplankton in the oceans across all latitudes were combinedto assess their community respiration on a global basis. Mesozooplanktonbiomass was higher in boreal/anti-boreal and polar waters, intermediatein equatorial waters and lowest in the subtropical gyres. Specificrespiration rates were the highest in equatorial waters anddecreased rapidly poleward. Global community respiration ofmesozooplankton in the upper 200 m of the oceans integratedover all latitudes was 10.4 ± 3.7 (SE) Gt C year^–1(n = 838). Below the epipelagic zone, mesozooplankton respirationliving in the mesopelagic (200–1000 m) and bathypelagic(below 1000 m) zones was estimated as 2.2 ± 0.4 (n =57) and 0.40 ± 0.2 (n = 12) Gt C year^–1, respectively.Thus, global depth-integrated mesozooplankton respiration was13.0 ± 4.2 Gt C year^–1 (17–32% of globalprimary production), which is 3–8-fold higher than thevalues assigned to mesozooplankton respiration in recent estimatesof total respiration in the ocean. Thus, it appears that mesozooplanktonrepresent a major, but neglected component of the carbon cyclein the ocean.     

Mobilization of Reserves and Synthesis of Sterols and Triterpenes in Seedlings of Two Canarian Euphorbia Species

Seedlings from Euphorbia canariensis and Euphorbia lambii weregrown in the dark at 25 °C. Protein and triglyceride contentas well as levels of sugars and amino acids in the endospermwere determined during endosperm depletion. In the endospermof Euphorbia canariensis, relatively low levels of amino acids(up to 1 µmol.endosperm^–1) were found of which glutamine/glutamateaccounted for 40% at the stage of radicle emergence. High levelsof amino acids (up to 4 µmol.endosperm^–1) comparedwith sugars (up to 2 µmol sucrose.endosperm^–1) weredetected in the endosperm of Euphorbia lambii. Arginine wasthe main component (28 µmol%) of the amino acids in thistissue. In both species amino acid composition changed graduallyduring endosperm depletion. Cotyledons retained their ability to absorb a variety of watersoluble substrates after removal of the endosperm. ¹⁴C from[U-¹⁴C]sucrose was effectively incorporated into the triterpenesof the laticifers and to a lesser extent into the sterols ofthe seedling. The highest incorporation values were found inyoung seedlings about 2 d after the emergence of the radicle.Seedlings of this age also showed high incorporation rates of¹⁴C from labelled alanine, serine, threonine, valine, leucineand isoleucine into both triterpenols and sterols, but no generalconclusions about metabolic channelling in lipid synthesis couldbe made. Endosperm, Euphorbia canariensis L. Euphorbia lambii Svent., sterols, triterpenols, amino acids, laticifer, biosynthesis     

Root Hydraulic Conductivity of Two Cactus Species in Relation to Root Age, Temperature, and Soil Water Status

The effects of root age, temperature, and soil water statuson root hydraulic conductivity (L_P) were investigated for twocactus species, Ferocactus acanthodes and Opuntia ficus-indica.The volumetric flux density of water was measured for excisedroot segments, either using negative hydrostatic pressures appliedto the proximal end or using reverse flow of water from theroot to the soil. For both species, L_P at 20 ?C increased withroot age, average values reaching a maximum of 3.9 ? 10^–7m s^–1 MPa^–1 for F. acanthodes and 5.2 ? 10^–7m s^–1 MPa^–1 for O.ficus-indica at 11 to 17 weeksof age; L_P subsequently declined with increasing root age forboth species. L_P was maximal at a temperature of about 10 ?Cfor the youngest roots (1–3 weeks), this optimum shiftingto 40 ?C for 8-week-old roots of both species. For older roots(up to 1.5-years-old), L_P increased with temperature from 0?C to 50 ?C, with a Q₁₀ of 1.3 between 20 ?C and 30 ?C. At asoil water potential (_soil) of –0.016 MPa, root L_P wasindependent of the direction of water flow for both species.Depending on root age, L_P declined 45- to 500-fold for F. acanthodesand 90- to 800-fold for O.ficus-indica as _soil was reduced from–0.016 to –1.06 MPa, consistent with a rectifier-likebehaviour with respect to water movement between soil and roots.Incorporation of such responses into water uptake models shouldlead to a better understanding of root function. Key words: Ferocactus acanthodes, Opuntia ficus-indica, water potential, tension, reverse flow     

Microzooplankton herbivory and bacterivory in Newfoundland coastal waters during spring, summer and winter

Grazing by microzooplankton on autotrophic and heterotrophicpicoplankton as well as >0.7 µm phytoplankton (as measuredby chlorophyll a) was quantified during July, August, October,January and April in the surface layer of Logy Bay, Newfoundland(47°38'14'N, 52°39'36'W). Rates of growth and grazingmortality of bacteria, Synechococcus and >0.7 µm phytoplanktonwere measured using the sea water dilution technique. Microzooplanktoningested 83–184, 96–366 and 64–118% of bacterial,Synechococcus and >0.7 µm phytoplankton daily potentialproduction, respectively and 34–111, 25–30 and 16–131%of bacterial, Synechococcus and >0.7 µm phytoplanktonstanding stocks, respectively. The trends in prey net growthrates followed the seasonal cycles of prey biomass, suggestingthat microzooplankton are important grazers in Newfoundlandcoastal waters. Ingestion was lowest during January and October(~2 µg C l^–1 day^–1) and highest in August(~20 µg C l^–1 day^–1). Aside from April when>0.7 µm phytoplankton represented the majority (~80%)of carbon ingested, bacterioplankton and <1 µm phytoplanktonrepresented most of the carbon ingested (~40–100%). Althoughmicrozooplankton have here-to-fore been unrecognized as an importantgrazer population in Newfoundland coastal waters, these resultssuggest that they play an important role in carbon flow withinthe pelagic food web, even at low temperatures in Logy Bay.     

Structural organization of the 3'' half of the rat thyroglobulin gene

We report the structural organization of an 80 Kb segment of rat DNA, which encodes for about 40% of Thyroglobulin mRNA at the 3' end. The codogenic information included in this segment is splitted in 17 exons of homogeneous size (about 200 bp). The seven exons at the extreme 3' end have been precisely defined by DNA sequence analysis. No clear sequence homology is found among the exons, even though their coding capacity is quite similar, from 55 to 63 aminoacids residues. We located 2 hormonogenic (T4 forming) sites on the extreme 3' end of the gene in different exons. The DNA sequence coding for these functional sites shows a 70% homology in a 50 nucleotides segment. In addition we found a remnant of this sequence in other exons of the gene. Two large introns have been found on the 3' end of the gene: one is 17 Kb and the other one is more than 30 Kb long. On the basis of these findings and of preliminary studies on the remaining 5' end of the gene, we can predict that the minimum length of the rat TGB gene will be 150 Kb, which makes this gene the largest so far identified eukaryotic gene. We propose in addition that the 3' end exons arose by duplication of a common ancestor.     

Structural Analysis of a recA-like Gene in the Genome of Arabidopsis thaliana

A recA-like gene was identified in the genome of Arabidopsisthaliana by means of PCR using primers designed on the basisof previously reported amino acid sequences of eukaryotic RecA-likeproteins. The structure of the gene, termed ArLIM15, was investigatedby comparing the primary structure of the genomic DNA with thatof the corresponding cDNA. The open reading frame, which wassplit into 15 exons, was established to have the capacity forencoding a 37.3-kDa polypeptide. The amino acid sequence ofthe putative product of ArLIM15 showed a high degree of similaritytothat of LIM15 in the monocotyledonous plant Lilium, includinga 93% identity, and to those of other recA-like genes in yeastsand vertebrates with identities of 69–71%. Phylogeneticanalysis indicated ArLIM15 to be much closer to meiosis-specificLIM15 and DMC1 in Saccharomyces cerevisiae than to RAD51 inS. cerevisiae and its homologues on an evolutionary scale.     

Laboratory and field observations on the scuticociliate Histiobalantium from the pelagic zone of Lake Constance, FRG

Histiobalantium sp. was found regularly in the pelagic zoneof Lake Constance, FRG, over five annual cycles. Maxima of upto 6400 cells l^–1 were recorded in late summer, with similarnumbers in the 0–8 and 8–20 m depth intervals. Onan annual average, the population accounted for 10–17%of the total biomass of planktonic ciliates. In the laboratory,Histiobalantium grew well on a diet of the cryptophyte Rhodomonassp. Maximum growth rates obtained in batch cultures were 0.21and 0.33 day^–11 at 9 and 18°C, respectively. In situexperiments using diffusion chambers yielded positive growthrates in autumn and winter. The highest values recorded at theambient temperatures 5, 14 and 17°C were 0.17, 0.32 and0.40 day^–1, respectively. Comparing these results withthe different seasonal distributions and higher measured growthrates of other ciliates from Lake Constance, we conclude thatHistiobalantium is a superior competitor at relatively low algalfood concentrations. ²Present address: Fisheries & Oceans Canada, 4160 MarineDrive, West Vancouver, BC, V7V 1N6, Canada