The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional over-expression

Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β–estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β–estradiol treatment. As a proof of concept for the exploitation of this resource, β–estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl–root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution.

Advanced morphometric techniques applied to the study of human brain anatomy

El desarrollo de las técnicas de imágenes por resonancia magnética han permitido el estudio y cuantificación, in vivo, de los cambios que ocurren en la morfología cerebral ligados a procesos tales como el neurodesarrollo, el envejecimiento, el aprendizaje o la enfermedad. Un gran número de métodos de morfometría han sido desarrollados con el fin de extraer la información contenida en estas imágenes y traducirla en indicadores de forma o tamaño, tales como el volumen o el grosor cortical; marcadores que son posteriormente empleados para encontrar diferencias estadísticas entre poblaciones de sujetos o realizar correlaciones entre la morfología cerebral y, por ejemplo, la edad o la severidad de determinada enfermedad. A pesar de la amplia variedad de biomarcadores y metodologías de morfometría, muchos estudios sesgan sus hipótesis, y con ello los resultados experimentales, al empleo de un número reducido de biomarcadores o a al uso de una única metodología de procesamiento. Con el presente trabajo se pretende demostrar la importancia del empleo de diversos métodos de morfometría para lograr una mejor caracterización del proceso que se desea estudiar. En el mismo se emplea el análisis de forma para detectar diferencias, tanto globales como locales, en la morfología del tálamo entre pacientes adolescentes con episodios tempranos de psicosis y adolescentes sanos. Los resultados obtenidos demuestran que la diferencia de volumen talámico entre ambas poblaciones de sujetos, previamente descrita en la literatura, se debe a una reducción del volumen de la región anterior-mediodorsal y del núcleo pulvinar del tálamo de los pacientes respecto a los sujetos sanos. Además, se describe el desarrollo de un estudio longitudinal, en sujetos sanos, que emplea simultáneamente distintos biomarcadores para la caracterización y cuantificación de los cambios que ocurren en la morfología de la corteza cerebral durante la adolescencia. A través de este estudio se revela que el proceso de “alisado” que experimenta la corteza cerebral durante la adolescencia es consecuencia de una disminución de la profundidad, ligada a un incremento en el ancho, de los surcos corticales. Finalmente, esta metodología es aplicada, en un diseño transversal, para el estudio de las causas que provocan el decrecimiento tanto del grosor cortical como del índice de girificación en adolescentes con episodios tempranos de psicosis. ABSTRACT The ever evolving sophistication of magnetic resonance image techniques continue to provide new tools to characterize and quantify, in vivo, brain morphologic changes related to neurodevelopment, senescence, learning or disease. The majority of morphometric methods extract shape or size descriptors such as volume, surface area, and cortical thickness from the MRI image. These morphological measurements are commonly entered in statistical analytic approaches for testing between-group differences or for correlations between the morphological measurement and other variables such as age, sex, or disease severity. A wide variety of morphological biomarkers are reported in the literature. Despite this wide range of potentially useful biomarkers and available morphometric methods, the hypotheses and findings of the grand majority of morphological studies are biased because reports assess only one morphometric feature and usually use only one image processing method. Throughout this dissertation biomarkers and image processing strategies are combined to provide innovative and useful morphometric tools for examining brain changes during neurodevelopment. Specifically, a shape analysis technique allowing for a fine-grained assessment of regional thalamic volume in early-onset psychosis patients and healthy comparison subjects is implemented. Results show that disease-related reductions in global thalamic volume, as previously described by other authors, could be particularly driven by a deficit in the anterior-mediodorsal and pulvinar thalamic regions in patients relative to healthy subjects. Furthermore, in healthy adolescents different cortical features are extracted and combined and their interdependency is assessed over time. This study attempts to extend current knowledge of normal brain development, specifically the largely unexplored relationship between changes of distinct cortical morphological measurements during adolescence. This study demonstrates that cortical flattening, present during adolescence, is produced by a combination of age-related increase in sulcal width and decrease in sulcal depth. Finally, this methodology is applied to a cross-sectional study, investigating the mechanisms underlying the decrease in cortical thickness and gyrification observed in psychotic patients with a disease onset during adolescence.

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Sex-specific quantitative trait loci affecting longevity in Drosophila melanogaster

Senescence, the decline in survivorship and fertility with increasing age, is a near-universal property of organisms. Senescence and limited lifespan are thought to arise because weak natural selection late in life allows the accumulation of mutations with deleterious late-age effects that are either neutral (the mutation accumulation hypothesis) or beneficial (the antagonistic pleiotropy hypothesis) early in life. Analyses of Drosophila spontaneous mutations, patterns of segregating variation and covariation, and lines selected for late-age fertility have implicated both classes of mutation in the evolution of aging, but neither their relative contributions nor the properties of individual loci that cause aging in nature are known. To begin to dissect the multiple genetic causes of quantitative variation in lifespan, we have conducted a genome-wide screen for quantitative trait loci (QTLs) affecting lifespan that segregate among a panel of recombinant inbred lines using a dense molecular marker map. Five autosomal QTLs were mapped by composite interval mapping and by sequential multiple marker analysis. The QTLs had large sex-specific effects on lifespan and age-specific effects on survivorship and mortality and mapped to the same regions as candidate genes with fertility, cellular aging, stress resistance and male-specific effects. Late age-of-onset QTL effects are consistent with the mutation accumulation hypothesis for the evolution of senescence, and sex-specific QTL effects suggest a novel mechanism for maintaining genetic variation for lifespan.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Telomere lengthening and telomerase activation during human B cell differentiation

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.

Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T2AG3)n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.

Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA

The primase DnaG of Escherichia coli requires the participation of the replicative helicase DnaB for optimal synthesis of primer RNA for lagging strand replication. However, previous studies had not determined whether the activation of the primase or its loading on the template was accomplished by a helicase-mediated structural alteration of the single-stranded DNA or by a direct physical interaction between the DnaB and the DnaG proteins. In this paper we present evidence supporting direct interaction between the two proteins. We have mapped the surfaces of interaction on both DnaG and DnaB and show further that mutations that reduce the physical interaction also cause a significant reduction in primer synthesis. Thus, the physical interaction reported here appears to be physiologically significant.

Perturbation of the T cell repertoire in rheumatoid arthritis

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) β-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR β-chain sequences of 1 in 2.4 × 107 CD4 T cells. In RA patients, the median TCR β-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P < 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P < 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.

Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate

Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase from Chenopodium album, screened the C. album cDNA library, and cloned a cDNA (CaCLH, C. album chlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop). CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) in Arabidopsis thaliana (AtCLH1). We expressed the gene products of AtCLH1 and of CaCLH in Escherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on an Arabidopsis genomic fragment and expressed it in E. coli, demonstrating the presence of the second Arabidopsis CLH gene (AtCLH2). No typical feature of signal sequence was identified in AtCLH1, whereas AtCLH2 had a typical signal sequence for chloroplast. AtCLH1 mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h. AtCLH2, however, did not respond to MeJA.

Loss of p21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in atm-deficient mice

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm-deficient mice recapitulate most of the AT phenotype. p21cip1/waf1 (p21 hereafter), an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to γ-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, we examined the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis. As might have been expected, our data provide evidence that p21 modifies the in vitro senescent response seen in AT fibroblasts. Further, it is a downstream effector of ATM-mediated growth control. In addition, however, we find that loss of p21 in the context of an atm-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular.

Cyclin A activates the DNA polymerase δ-dependent elongation machinery in vitro: A parvovirus DNA replication model

Replication of the single-stranded linear DNA genome of parvovirus minute virus of mice (MVM) starts with complementary strand synthesis from the 3′-terminal snap-back telomere, which serves as a primer for the formation of double-stranded replicative form (RF) DNA. This DNA elongation reaction, designated conversion, is exclusively dependent on cellular factors. In cell extracts, we found that complementary strand synthesis was inhibited by the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and rescued by the addition of proliferating cell nuclear antigen, arguing for the involvement of DNA polymerase (Pol) δ in the conversion reaction. In vivo time course analyses using synchronized MVM-infected A9 cells allowed initial detection of MVM RF DNA at the G1/S phase transition, coinciding with the onset of cyclin A expression and cyclin A-associated kinase activity. Under in vitro conditions, formation of RF DNA was efficiently supported by A9 S cell extracts, but only marginally by G1 cell extracts. Addition of recombinant cyclin A stimulated DNA conversion in G1 cell extracts, and correlated with a concomitant increase in cyclin A-associated kinase activity. Conversely, a specific antibody neutralizing cyclin A-dependent kinase activity, abolished the capacity of S cell extracts for DNA conversion. We found no evidence for the involvement of cyclin E in the regulation of the conversion reaction. We conclude that cyclin A is necessary for activation of complementary strand synthesis, which we propose as a model reaction to study the cell cycle regulation of the Pol δ-dependent elongation machinery.

The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family

The plant hormone ethylene is involved in many developmental processes, including fruit ripening, abscission, senescence, and leaf epinasty. Tomato contains a family of ethylene receptors, designated LeETR1, LeETR2, NR, LeETR4, and LeETR5, with homology to the Arabidopsis ETR1 ethylene receptor. Transgenic plants with reduced LeETR4 gene expression display multiple symptoms of extreme ethylene sensitivity, including severe epinasty, enhanced flower senescence, and accelerated fruit ripening. Therefore, LeETR4 is a negative regulator of ethylene responses. Reduced expression of this single gene affects multiple developmental processes in tomato, whereas in Arabidopsis multiple ethylene receptors must be inactivated to increase ethylene response. Transgenic lines with reduced NR mRNA levels exhibit normal ethylene sensitivity but elevated levels of LeETR4 mRNA, indicating a functional compensation of LeETR4 for reduced NR expression. Overexpression of NR in lines with lowered LeETR4 gene expression eliminates the ethylene-sensitive phenotype, indicating that despite marked differences in structure these ethylene receptors are functionally redundant.

Altered recognition mutants of the response regulator PhoB: A new genetic strategy for studying protein–protein interactions

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a “patch” near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and Pi signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6Kγ attP “genome targeting” suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.