Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene

The mouse pre-T-cell receptor alpha (pT) chain is a 33 000 M _r glycoprotein expressed on the surface of immature thymocytes as a disulfide-linked heterodimer with the T-cell receptor beta (TCR) chain, and in association with proteins of the CD3 complex. The cDNA for pT, isolated previously, encodes a type I transmembrane protein that is a member of the immunoglobulin (Ig) superfamily. Here we report the complete nucleotide sequence, the exon/intron structure, and the chromosomal location of the pTa gene. The gene spans about 8.4 kilobases (kb) and consists of four exons. Exon 1 encodes the 5 untranslated region, the leader peptide, and the first three amino acids of the mature protein. This exon is followed by a relatively long intron of 4.9 kb that contains many short interspersed repeats (SINEs) of the B1 and B2 family. The second exon encodes the extracellular Ig-like domain and exon 3 with just 45 base pairs the connecting peptide (CP), including the cysteine required for heterodimer formation. A similar exon/intron structure encoding corresponding parts of the mature polypeptide is found both in the Tcra and Tcrd constant region genes. The last exon encodes the transmembrane portion, the cytoplasmic tail, and about 540 nucleotides of 3 untranslated sequence, including a B2 repetitive element. In situ hybridization maps the pTa gene to the D/E1 region of mouse chromosome 17.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U27268     

Toxin A secretion in Pseudomonas aeruginosa: the role of the first 30 amino acids of the mature toxin

Toxin A, one of several virulence factors secreted by the gram-negative bacterium Pseudomonas aeruginosa, is synthesized as a 71 kDa precursor with a typical prokaryotic leader peptide (LP), and is secreted as a 68 kDa mature protein. Evidence from a previous study suggested that a signal required for toxin A secretion in P. aeruginosa may reside within the region defined by the toxin A LP and the first 30 amino acids (aa) of mature toxin A. In the present study, we have used exonuclease Ba131 deletion analysis to examine the specific role of the first 30 as in toxin A secretion. Four toxA subclones, which encode products containing the toxin A LP and different segments of the 30-residue region fused to a toxin A carboxy-terminal region, were identified. In addition, a gene fusion encoding a hybrid protein consisting of the LP of P. aeruginosa elastase and the final 305 residues of toxin A, was generated. The cellular location of the toxA subclone products in P. aeruginosa was determined by immunoblotting analysis. Toxin A CRMs (cross-reacting material) encoded by different subclones were detected in different fractions of P. aeruginosa including the periplasm and the supernatant. Results from these studies suggest that (1) mature toxin A contains two separate secretion signals one within the N-terminal region and one within the C-terminal region; and (2) the first 30 residues of the mature toxin A form part of the N-terminal secretion signal.     

Effect of DNA fragment size on transformation frequencies in tobacco (Nicotiana tabacum) and maize (Zea mays)

During eukaryotic cell transformation, the transforming DNA must enter the host cell, traverse the cytoplasm and enter the nucleus before becoming stably integrated into the genome. The limiting step for plant protoplast transformation may lie at the cell membrane, the nuclear membrane, or at the integration step. We show here that the size of the DNA fragment containing the selectable marker used to monitor transformation can directly affect the efficiency of stable transformation. In both tobacco and maize protoplasts, the smallest DNA fragments gave the highest stable transformation frequencies.     

The F3 Neuronal Glycosylphosphatidylinositol-Linked Molecule Is Localized to Glycolipid-Enriched Membrane Subdomains and Interacts with L1 and Fyn Kinase in Cerebellum

Abstract: The F3 molecule is a member of the immunoglobulin superfamily anchored to plasma membranes by a glycosylphosphatidylinositol group. In adult mouse cerebellum, F3 is predominantly expressed on a subset of axons, the parallel fibers, and at their synapses. In vitro studies established that it is a plurifunctional molecule that, depending on the cellular context and the ligand with which it interacts, either mediates repulsive interactions or promotes neurite outgrowth. In the present study, we report the isolation of two fractions of F3-containing microdomains from adult cerebellum on the basis of their resistance to solubilization by Triton X-100 at 4°C. Both fractions were composed of vesicles, ranging from 100 to 200 nm in diameter. Lipid composition analysis indicated that the lighter fraction was enriched in cerebrosides and sulfatides. F3 sensitivity to phosphatidylinositol phospholipase C differed between the two fractions, possibly reflecting structural differences in the lipid anchor of the F3 molecule. Both fractions were highly enriched in other glycosylphosphatidylinositol-anchored proteins such as NCAM 120 and Thy-1. It is interesting that these vesicles were devoid of the transmembrane forms (NCAM 180 and NCAM 140), which were recovered in Triton X-100-soluble fractions, but contained the L1 transmembrane adhesion molecule that is coexpressed with F3 on parallel fibers and the fyn tyrosine kinase. Immunoprecipitation experiments indicated that F3, but not NCAM 120 or Thy-1, was physically associated in a complex with both L1 and fyn tyrosine kinase. This strongly suggests that the interaction between L1 and F3, already described to occur with isolated molecules, is present in neural tissue. More important is that our study provides information on the molecular machinery likely to be involved in F3 signaling.     

The transposable element mariner can excise in non-drosophilid insects

Plasmid-based excision assays performed in embryos of two non-drosophilid species using the mariner transposable element from Drosophila mauritiana resulted in empty excision sites identical to those observed after the excision of mariner from D. mauritiana chromosomes. In the presence of the autonomous mariner element Mos1, excision products were recovered from D. melanogaster, D. mauritiana and the blowfly Lucilia cuprina. When a hsp82 heat shock promoter-Mos1 construct was used to supply mariner transposase, excision products were also recovered from the Queensland fruitfly Bactrocera tryoni. Analysis of DNA sequences at empty excision sites led us to hypothesise that the mariner excision/repair process involves the formation of a heteroduplex at the excision breakpoint. The success of these assays suggests that they will provide a valuable tool for assessing the ability of mariner and mariner-like elements to function in non-drosophilid insects and for investigating the basic mechanisms of mariner excision and repair.     

Transgenic and knockout rodents: Novel insights into mechanisms of body weight regulation

Transgenic animals that over- or underexpress a protein of interest have been used to study obesity development, prevention, and susceptibility to diet-induced obesity such as a high-fat diet. Several transgenic models are resistant to diet-induced obesity including those that overexpress the insulin-sensitive glucose transporter, GLUT4, in adipose tissue only. In this animal there is increased adipose tissue mass but the animal maintains its insulin sensitivity. The overexpression of lipoprotein lipase (LPL) in skeletal muscle and the elimination of a protein kinase A subunit both resulted in lean and obesity resistant animals. By directing the production of the diphtheria toxin A chain to adipose tissue only the resulting animals not only had less adipose tissue mass but were resistant to MSG-induced obesity. Conversely, transgenic models with decreased brown adipose tissue or its function have all resulted in obese animals, highlighting the importance of thermoregulation in body weight maintenance. The use of transgenic technology in the field of obesity has emphasized the regional differences among fat pads as well as the dissimilarity between genders in fuel metabolism. Several transgenic models have separated obesity from insulin resistance allowing the importance of each state to be studied individually. Results using transgenic animals have re-emphasized that obesity is a polygenic disease.     

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.