Gene function and expression level influence the insertion/fixation dynamics of distinct transposon families in mammalian introns

Background  

Transposable elements (TEs) represent more than 45% of the human and mouse genomes. Both parasitic and mutualistic features have been shown to apply to the host-TE relationship but a comprehensive scenario of the forces driving TE fixation within mammalian genes is still missing.     

Introns in,introns out in plant gene families: a genomic approach of the dynamics of gene structure

Gene duplication is considered to be a source of genetic information for the creation of new functions. The Arabidopsis thaliana genome sequence revealed that a majority of plant genes belong to gene families. Regarding the problem of genes involved in the genesis of novel organs or functions during evolution, the reconstitution of the evolutionary history of gene families is of critical importance. A comparison of the intron/exon gene structure may provide clues for the understanding of the evolutionary mechanisms underlying the genesis of gene families. An extensive study of A. thaliana genome showed that families of duplicated genes may be organized according to the number and/or density of intron and the diversity in gene structure. In this paper, we propose a genomic classification of several A. thaliana gene families based on introns in an evolutionary perspective. abbreviations BGAL, -galactosidases; PCMP, plant combinatorial and modular protein     

Gene function in the mammalian genome,courtesy of the mouse

A report on the 16th International Mouse Genome Conference, San Antonio, USA, 17-20 November, 2002.     

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: effect of a transposon insertion in the hbdA gene

The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-beta-hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.     

A murine gene with circadian expression revealed by transposon insertion: self-sustained rhythmicity in the liver and the photoreceptors

We have previously identified in some mouse strains (e.g. BALB/c, DBA/2) a murine Intracisternal A-particle (IAP) transposable element specifically expressed in the liver. This IAP sequence is inserted within a gene, mCCR4/m. nocturnin, the sequence of which is related to the circadian Xenopus nocturnin gene. Here we show, using real-time quantitative RT-PCR, that both the IAP sequence and the m. nocturnin gene display strong circadian expression in the liver, with peak abundance after dusk. Circadian oscillations of m. nocturnin RNA are maintained in mice without the IAP insertion (e.g. CBA/J, 129/sv), are free-running under constant light and dark conditions, and persist upon food and water privation, demonstrating that m. nocturnin is a circadian gene. In situ hybridization analyses (in 129/sv mice) further show circadian expression of m. nocturnin also in the retina, precisely at the level of the photoreceptors, a result consistent with the previously described circadian expression of the Xenopus gene. These results strengthen the strong conservation of the nocturnin gene with the identification of a functional mouse ortholog of the Xenopus gene, and demonstrate the reciprocal influence of nearby genes on the expression of transposable elements via "position effects".     

Diversity of glutamate transporter expression and function in the mammalian retina

Summary. Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. Between photoreceptors and second order neurons, increases in light intensity are signaled by decreases in the concentration of glutamate within the synaptic cleft. In such a system the precise control of glutamate in the synaptic cleft is thus essential and glutamate transporters are thought to contribute to this process. As demonstrated here, all neuronal and macroglial cells of the retina appear to express high-affinity glutamate transporters. GLAST1, GLT1, EAAC1 and EAAT5 are expressed in the retina and exhibit unique localisation and functional properties. In the present study we summarize retinal glutamate transporter expression, identify the major glutamate uptake site in the mammalian retina and discuss the possible functional roles of different glutamate transporter subtypes in glutamatergic neurotranmission in the retina. Received August 31, 1999 Accepted September 20, 1999     

Phenotypic manifestations of yeast transposon insertion in the LYS2 gene and deletions resulting from imprecise excision of the transposon

The lys2-32 mutant allele resulted from Ty1 element insertion was identified and cloned. The expression and reversions of lys2-32 localized in an autonomous plasmid were studied. The insertion was shown to inactivate LYS2 gene incompletely. Spontaneous reversions to complete or almost complete prototrophy were also obtained. About 50% of revertants retained the insertion. Others arise as a result of imprecise excision events leading to deletions of adjacent LYS2 sequences.     

An analysis of the factors that influence the level and scaling of mammalian BMR

The factors influencing the basal rate of metabolism (BMR) in 639 species of mammals include body mass, food habits, climate, habitat, substrate, a restriction to islands or highlands, use of torpor, and type of reproduction. They collectively account for 98.8% of the variation in mammalian BMR, but often interact in complex ways. The factor with the greatest impact on BMR, as always, is body mass (accounting for 96.8% of its variation), the extent of its impact reflecting the 10(6.17)-fold range of mass in measured species. The attempt to derive mathematically the power relationship of BMR in mammals is complicated by the necessity to include all of the factors that influence BMR that are themselves correlated with body mass. BMR also correlates with taxonomic affiliation because many taxa are distinguished by their ecological and behavioral characteristics. Phylogeny, reflecting previous commitments, may influence BMR either through a restriction on the realized range of behaviors or by opening new behavioral and ecological opportunities. A new opportunity resulted from the evolution by eutherians of a type of reproduction that permitted species feeding on high quality resources to have high BMRs. These rates facilitated high rates of gas, nutrient, and waste exchange between a pregnant eutherian and her placental offspring. This pattern led to high rates of reproduction in some eutherians, a response denied all monotremes and marsupials, thereby permitting eutherians to occupy cold-temperate and polar environments and to dominate other mammals in all environments to which ecologically equivalent eutherians had access.     

Random insertion of the gentamicin resistance transposon Tn4001 in Mycoplasma pulmonis

The staphylococcal transposon Tn4001 was introduced into Mycoplasma pulmonis using an Escherichia coli-derived vector by polyethylene glycol-mediated transformation. Using a reaction mixture containing 10 micrograms plasmid DNA, 10 micrograms yeast tRNA, and 34-35% polyethylene glycol per 1 x 10(8) cells, Tn4001 could be introduced into M. pulmonis at a frequency of 5 x 10(-5) per colony forming unit. DNA-DNA hybridization studies illustrated that Tn4001 could occupy a diversity of insertion sites in the M. pulmonis chromosome. These data indicated that Tn4001 is a potentially useful tool for the introduction of mutations and for genetic studies in M. pulmonis.     

Prions: on the enzymatic function of PrPc from mammalian and prion "families"

This minireview deals with some approaches and results of experiments which enabled to discover SOD-like activity of the mammal PrP protein. This activity required the unchanged region of the repeated octapeptide and Cu2+ binding to the appropriate sites of the PrP molecule. It was shown that an infectious prion isoform could bind the normal isoform. This leads to the loss of PrP SOD-like function accompanied by Cu2+ release from the molecule. Also, the problem of sowing the protein seeds of prion propagation is briefly summarized and the first evidence of abnormal protein conformation induced in vivo using yeast cell and in vitro formed Sup 35 prion seed is described.     

The function of the small insertion in the hinge subdomain in the control of constitutive mammalian nitric-oxide synthases

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.     

Evidence for the importance of electrostatics in the function of two distinct families of ribosome inactivating toxins

Alpha-sarcin and ricin represent two structurally and mechanistically distinct families of site-specific enzymes that block translation by irreversibly modifying the sarcin/ricin loop (SRL) of 23S-28S rRNA. alpha-Sarcin family enzymes are designated as ribotoxins and act as endonucleases. Ricin family enzymes are designated as ribosome inactivating proteins (RIP) and act as N-glycosidases. Recently, we demonstrated that basic surface residues of the ribotoxin restrictocin promote rapid and specific ribosome targeting by this endonuclease. Here, we report that three RIP: ricin A, saporin, and gypsophilin depurinate the ribosome with strong salt sensitivity and achieve unusually fast kcat/Km approximately 10(9)-10(10) M(-1) s(-1), implying that RIP share with ribotoxins a common mechanism of electrostatically facilitated ribosome targeting. Bioinformatics analysis of RIP revealed that surface charge properties correlate with the presence of the transport chain in the RIP molecule, suggesting a second role for the surface charge in RIP transport. These findings put forward surface electrostatics as an important determinant of RIP activity.     

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.