Feed form and energy concentration of the diet affect growth performance and digestive tract traits of brown-egg laying pullets from hatching to 17 weeks of age

The influence of feed form and energy concentration of the diet on growth performance and the development of the gastrointestinal tract (GIT) was studied in brown-egg laying pullets. Diets formed a 2 x 5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of energy differing in 50 kcal AMEn/kg. For the entire study (0 to 17 wk of age) feeding crumbles increased ADFI (52.9 vs. 49.7 g; P < 0.001) and ADG (12.7 vs. 11.6 g; P < 0.001) and improved feed conversion ratio (FCR; 4.18 vs. 4.27; P < 0.001). An increase in the energy content of the diet decreased ADFI linearly (P < 0.001) and improved FCR quadratically (P < 0.01) but energy intake (kcal AMEn/d) was not affected. BW uniformity was higher (P < 0.05) in pullets fed crumbles than in those fed mash but was not affected (P > 0.05) by energy content of the diet. At 5, 10, and 17 wk of age, the relative weight (RW, % BW) of the GIT and the gizzard, and gizzard digesta content were lower (P < 0.05 to P < 0.001) and gizzard pH was higher (P < 0.05 to P < 0.001) in pullets fed crumbles than in pullets fed mash. Energy concentration of the diet did not affect any of the GIT variables studied. In summary, feeding crumbles improved pullet performance and reduced the RW of the GIT and gizzard, and increased gizzard pH at all ages. An increase in the energy content of the diet improved FCR from 0 to 17 wk of age. The use of crumbles and the increase in the AMEn content of the diet might be used adventageously when the objetive is to increase the BW of the pullets. However, crumbles affected the development and weight of the organs of the GIT, which might have negative effects on feed intake and egg production at the beginning of the egg laying cycle.

Influence of the main cereal and feed form of the rearing phase diets on performance, digestive tract, and body traits of brown-egg laying pullets from hatch to 17 weeks of age

We hypothesize that pullets could respond similarly, independent of feed form, to the feeding of diets based on corn or wheat supplemented with adequate NSP enzymes. Also, pullets would quickly adapt their gastrointestinal tract and modify productive performance accordingly, when switched from crumbles to mash feeds. The aim of this research was to evaluate the effects of feeding crumbles for different periods of time, followed by feeding mash to 17 wk of age, on performance, gastrointestinal tract development, and body measurements of brown-egg laying pullets fed diets based on corn or wheat.

Some examples of the simultaneous use of closed-form and numerical solutions in shell structural analysis

The computational advantages of the use of different approaches -numerical and analytical ones- to the analysis of different parts of the same shell structure are discussed. Examples of large size problems that can be reduced to those more suitable to be handled by a personal related axisyrometric finite elements, local unaxisymmetric shells, geometric quasi-regular shells, infinite elements and homogenization techniques are described

Productive performance of brown-egg laying pullets from hatching to 5 weeks of age as affected by fiber inclusion, feed form, and energy concentration of the diet1

The effects of fiber inclusion, feed form, and energy concentration of the diet on the growth performance of pullets from hatching to 5 wk age were studied in 2 experiments. In Experiment 1, there was a control diet based on cereals and soybean meal, and 6 extra diets that included 2 or 4% of cereal straw, sugar beet pulp (SBP), or sunflower hulls (SFHs) at the expense (wt/wt) of the whole control diet. From hatching to 5 wk age fiber inclusion increased (P < 0.05) ADG and ADFI, and improved (P < 0.05) energy efficiency (EnE; kcal AMEn/g ADG), but body weight (BW) uniformity was not affected. Pullets fed SFH tended to have higher ADG than pullets fed SBP (P = 0.072) with pullets fed straw being intermediate. The feed conversion ratio (FCR) was better (P < 0.05) with 2% than with 4% fiber inclusion. In Experiment 2, 10 diets were arranged as a 2×5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of AMEn (2,850, 2,900, 2,950, 3,000, and 3,050 kcal/kg). Pullets fed crumbles were heavier and had better FCR than pullets fed mash (P < 0.001). An increase in the energy content of the crumble diets reduced ADFI and improved FCR linearly, but no effects were detected with the mash diets (P < 0.01 and P < 0.05 for the interactions). Feeding crumbles tended to improve BW uniformity at 5 wk age (P = 0.077) but no effects were detected with increases in energy concentration of the diet. In summary, the inclusion of moderate amounts of fiber in the diet improves pullet performance from hatching to 5 wk age. The response of pullets to increases in energy content of the diet depends on feed form with a decrease in feed intake when fed crumbles but no changes when fed mash. Feeding crumbles might be preferred to feeding mash in pullets from hatching to 5 wk age.

Capilla del M.I.T. de Eero Saarinen, 1950-55 : sincretismo en la armonía de los opuestos

La historia de la arquitectura se ha medido - en parte - por la evolución de las modas, épocas y estilos de la arquitectura religiosa. En la aparición de las nuevas capillas interreligiosas universitarias de los años '50 de Norteamérica, la capilla del M.I.T., bautizada tras su inauguración como “Kresge Chapel” en honor al apellido de su benefactor, fue el máximo exponente como prototipo en esta época marcada por la aparición nuevas conciencias de posguerra. (II Guerra Mundial). La tesis centra su investigación en esta capilla, cómo nació en respuesta a la necesidad de compatibilizar la enseñanza reglada científico tecnológica con una formación religiosa y humanista, para el programa impuesto como ampliación del campus universitario del MIT en el barrio universitario de Cambridge, Boston, Massachussets, en torno a dos edificios principales a proyectar: capilla y auditorio. Desde el trabajo de investigación se aporta la información documental necesaria para saber cómo evolucionó desde sus primeros bocetos hasta su construcción. Su nacimiento como necesidad en la sociedad universitaria no fue nada espontáneo, quedando a medias entre la influencia escandinavo - alemana y la tradición americano-luterana de anteriores iglesias neoclásicas herederas del “Plan Akron”. La formación académica y profesional dirigida por su padre, Eliel Saarinen, suman junto a los viajes del arquitecto, una herencia “genética” para con este prototipo ajeno al emergente “estilo internacional” y ayudan a comprender la dimensión compleja de lo que es capaz de representar la capilla. La capilla es refugio emocional de la luz y es un punto de inflexión notable en la recuperación del tipo centrado renovado y evolucionado, que junto con el doble recurso lumínico - efectista, de la vertical para el altar y horizontal inferior, distribuido desde el perímetro ondulado en el interior, el ejemplo es muestra la promoción de una cierta sensibilidad para con las nuevas formas de la "religión" emergentes en la Norteamérica de los años 50. Desde el sincretismo como mecanismo y principal “modus operandi” proyectual, FE versus RAZÓN, aparecen como dualismo permanente en todas las fases del proyecto, desde un proceso de búsqueda de la armonía de ambos y para cada uno de ellos. Con el estudio del modelo prototípico del MIT, buscamos el "patrón" empleado por el arquitecto en su proyección de "nueva iglesia" adaptado a la diversidad cultural y religiosa y de las nuevas y diferentes sensibilidades humanistas para el proyecto. ----------------------------------------------------- SUMMARY--------------------------------------------------- The history of architecture is measured - in part - by the evolution of fashion, periods and styles of religious architecture. In the emergence of the new interfaith university chapel of the 1950s in North America, the chapel of MIT, named after its inauguration as "Kresge Chapel" in honor of its benefactor, was the best example of a prototype, in this time so marked by the emergence of new postwar consciousnesses. The thesis focuses its research on this chapel; how it came about in response to the need to reconcile formal scientific and technological education with religious and humanist training for the program imposed as an extension of the campus of MIT in the university district of Cambridge, Boston Massachusetts, around two main projected buildings : Chapel and auditorium. From research work has been obtained the documentary information necessary to know how it evolved from the first sketches to its construction. Its creation as a necessity of the university society was not at all spontaneous, being halfway between the Scandinavian influence - German and the American Lutheran tradition of neoclassical churches, the heirs to the "Akron Plan". The academic and professional training directed by his father, Eliel Saarinen, together with the travels of the architect, a "genetic" heritage with this external prototype for the emerging "international style", and help to understand the complex dimension of what the chapel is capable of representing. The chapel is an emotional refuge of light and is a remarkable turning point in the recovery of such focused renewed and evolved, along with the double lumen resource - gimmicky, vertical to the altar and lower horizontal, distributed from the undulating perimeter inside, the example is shown to promote a certain sensitivity to the new forms of "religion" but vague religious profile emerging in the America of the 50s. From the syncretism as a mechanism and main "modus operandi" of the project, FAITH versus REASON appears as permanent dualism in all phases of the project from a process of finding the harmony of both and for each of them. With the study of the prototypical model of MIT, we seek the "pattern" used by the architect in his projection of "new church" adapted to the cultural and religious diversity and new and different humanist sensitivities for the project. El desarrollo vierte la luz suficiente para entender los patrones de lo arquitectónico en la capilla, que hacen de ella, la comunión armónica y perfecta de los nombrados opuestos en el campus universitario del MIT. En el camino, además, la investigación arroja luz y orden sobre las fases del proyecto a través de la contribución, recogida, clasificación y orden de los datos, fechas y documentos gráficos, a día de hoy dispersos y confusamente publicados, debido a la no existencia de un estudio profundo y completo como el que pretende ser este trabajo, por ser una obra fundamental en la historia de la arquitectura moderna. El patrón final demostrará el artificio sincrético de cada una de las partes - cada uno en sí misma y cosidas todas - formando el "Ima Summis" de la capilla; resultado de la acción proyectual deducida de su serie genética descifrada. De esta forma, lo sincrético, aparece como el principal atributo del hecho construido, pasando de lo místico a lo científico, de lo intuitivo a lo razonado y siempre con el vehículo de su arquitectura para la interpretación de lo inefable al interior. En cuanto a la estructura de la tesis, se inicia el desarrollo a partir de una introducción en la que se declaran las intenciones y se describe el contexto de lo investigado en torno a la hipótesis principal anunciada en el subtítulo de la tesis. Frente a consideraciones previas de la capilla y de la propia investigación, se expone y explica la hipótesis principal, junto a otros objetivos secundarios. Para finalizar la introducción, se describe el método seguido como estrategia y se justifica la estructura de redacción del documento para la compresión de este trabajo hacia sus conclusiones. Es en el primer capítulo, el C1, donde se inicia el cuerpo central mediante la exposición historiográfica de la situación y contexto previo a la capilla como antecedentes. En un segundo capítulo, el C2, se aborda el estudio del desarrollo del proyecto y de la obra y construcción de la capilla, base documental necesaria. Para ello se inicia con la descripción del lugar, del encargo y del programa, para pasar a mostrar con detalle las distintas propuestas de cada una de las fases del proyecto. En definitiva, qué se proyecto, cómo evolucionó el proyecto y en qué fases transcurrió, para entre otras cuestiones, entender el cambio de estilo desde una fase influenciada por la capilla de Mies construida en el ITT de Chicago, (denominadas en este trabajo por esta influencia como modelos “miesianos”) y la fase reencuentro con el modus de hacer iniciado por su padre Eliel, a través de un modelo a medias de una herencia escandinavo - alemana y la tradición luterano - americana, (denominada en este trabajo como herencia o modelos “saariniana/os”). The development sheds enough light to understand the architectural patterns of the chapel, making it the perfect and harmonic communion named opposites on the campus of MIT. Along the way, furthermore, the research sheds light and order on the phases of the project through the contribution, collection, sorting and order of data, dates and graphic documents, today scattered and confusingly published, due to inexistence of a deep and comprehensive study like this work is meant to be, in the context of such a seminal work in the history of modern architecture. The final pattern will demonstrate the syncretic artifice of each of the parts - each in itself and all together - forming the "Ima Summis" of the chapel; the result of the project action deducted from its deciphered genetic series. Being the SYNCRETIC, the main attribute of the built matter, passing from the mystical to the scientific, from the intuitive to the reasoned and always with the vehicle of architecture as the representation of the ineffable. As for the structure of the thesis, the development starts from an introduction in which the intentions are declared and the context of that which is investigated is described, in terms of the main hypothesis announced in the subtitle of the thesis. Faced with previous considerations of the chapel and the research itself, the principal hypothesis is expressed and explained, along with other secondary objectives. To conclude the introduction, the method followed is described as a strategy and the structuring of the document is justified for the compression of this work towards its conclusions. It is in the first chapter of the thesis, the C1, where the main body of the work is initiated through the historiographical account of the situation and context prior to the chapel as antecedents. A second chapter, C2, addresses the study of the development of the project and of the work and construction of the chapel, based on the necessary documentary evidence. To this end, the analysis begins with the description of the place, the commission and the program, moving on to analyze in detail the various proposals of each phase. What is projected, how it evolved and through what phases passed the project to, among other questions, understand the change in style between the phase of influence of Mies through the projected chapel in the ITT of Chicago chapel (called in this work as "miesian" influences or models) and the reunion with the modus of making initiated by his father Eliel, through a model equally at once of Scandinavian-German heritage and of the American-Lutheran tradition, (referred to in this work as "saarinian" inheritance or models). Para el tercer capítulo, el C3, se llevan a estudio y análisis, los elementos más destacables en los que se prueban las particularidades llamadas “genéticas” en cada uno de las partes principales detectadas en la capilla. Desde ellas se rastrean las influencias - a veces sinergias - que ayudan a justificar y conformar una clasificación genética en torno a cuatro patrones principales, de los que poder discernir al final del capítulo, el “patrón matriz” de la propia capilla en base a la concurrencia de los órdenes arquitectónicos que los conforman. Una vez obtenida la información y orden necesario, se puede afrontar el punto álgido de esta tesis con el cuarto capítulo, el C4, en el que se justifica la capacidad sincrética de la capilla como respuesta a la hipótesis principal señalada en el subtítulo. Para ello se inicia el capítulo con el enfoque y contexto del término “sincretismo” y “sincrético”, aportando la justificación de sus usos para con esta investigación, antes de explicar que la capilla es sincrética por la suma de dos claves fundamentales. La clave primera, correspondiente a la capacidad interconfesional y aconfesional de la capilla, y su capacidad de trascender al interior desde ambas situaciones además de ser reconocible por el religioso y a la vez por el científico. Y la clave segunda, que corresponde a la suma de las distintas partes sincréticas, cada una en si mismas, capaz de sumar todas juntas, este edificio sincrético de los nombrados opuestos. Una vez discernido las claves de lo sincrético en la capilla y a pesar de su doble carácter SACRO Y PROFANO, una comparativa posiciona a la capilla como único entre los ejemplos interconfesionales construidos en los ´50, sin haberse repetido de igual manera y mismo resultado en el tiempo, ni en ninguna otra universidad o centro tecnológico. Así la exposición de este trabajo finaliza con el último capítulo, el C5, en el que desde esa comparativa, una serie de cuestiones unen y distancian a la del MIT y dan acierto a la apuesta inicial, concluyendo que la hipótesis y el patrón de la misma, es el patrón de lo sincrético que modela el ”modus operandi ” del arquitecto. Tras el desarrollo, la investigación cierra filas en torno a cuatro conclusiones para los cuatro capítulos principales; C2, C3, C4, C5. La capilla del MIT como modelo prototípico no se repite en el tiempo, ni en ninguna otra universidad y se posiciona como el mejor prototipo – hito de una nueva arquitectura religiosa universitaria. In the third chapter, C3, we study and analyze the most notable elements in which are found those particularities referred to as "genetic" in each of the principal parts, (see above C3.1.1... - C3.1.8), where these influences - sometimes synergies - are traced, helping to justify and form a classification based on four of those principals (see above C3.2), from which, at the end of this chapter may be discerned a matrix pattern, that of the chapel itself (see above. C3.3.), based on concurrent architectural orders. Once the necessary information and order is obtained, we come to the decisive point in the fourth chapter, C4, where the syncretic capacity of the chapel in response to the main hypothesis indicated in the subtitle of the thesis is justified. For this, the chapter beings with the focus and context of the terms "syncretism" and "syncretic", providing justification for their use for this research, going on to assert that the chapel is syncretic from the sum of two fundamental aspects: the first corresponds to the interfaith and non-denominational capacities of the chapel, its ability to transcend both situations and be recognizable by both the religious and the scientific. The second corresponds to the sum of the different syncretic parts, each in themselves, capable of making together a syncretic building with the opposite. Once discerned the keys to the syncretic and epic in the chapel and despite its dual character both SACRED and PROFANE, a comparatison positions the chapel as being unique among interfaith examples built in the 1950s, without being repeated in the same way and with the same result over time or in any other university or centre of technology. So, the exhibition of this work ends with the last chapter, C5, in which from that comparison a number of issues come together and to distance themselves from MIT and bear out the initial proposition, concluding that the hypothesis and its pattern, the pattern of the syncretic which models the "modus operandi" of the architect to contain the ineffable. The research closes ranks around four conclusions for the four main chapters; C2, C3, C4, and C5 The chapel of MIT as a prototypical model is not repeated in time, or any other university and is positioned as the best prototype - a landmark of a new universitary religious architecture.

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

The N-terminal domain of PsaF: Precise recognition site for binding and fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii

The PsaF-deficient mutant 3bF of Chlamydomonas reinhardtii was used to modify PsaF by nuclear transformation and site-directed mutagenesis. Four lysine residues in the N-terminal domain of PsaF, which have been postulated to form the positively charged face of a putative amphipathic α-helical structure were altered to K12P, K16Q, K23Q, and K30Q. The interactions between plastocyanin (pc) or cytochrome c6 (cyt c6) and photosystem I (PSI) isolated from wild type and the different mutants were analyzed using crosslinking techniques and flash absorption spectroscopy. The K23Q change drastically affected crosslinking of pc to PSI and electron transfer from pc and cyt c6 to PSI. The corresponding second order rate constants for binding of pc and cyt c6 were reduced by a factor of 13 and 7, respectively. Smaller effects were observed for mutations K16Q and K30Q, whereas in K12P the binding was not changed relative to wild type. None of the mutations affected the half-life of the microsecond electron transfer performed within the intermolecular complex between the donors and PSI. The fact that these single amino acid changes within the N-terminal domain of PsaF have different effects on the electron transfer rate constants and dissociation constants for both electron donors suggests the existence of a rather precise recognition site for pc and cyt c6 that leads to the stabilization of the final electron transfer complex through electrostatic interactions.

Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I

Hereditary tyrosinemia type I (HT1) is an autosomal recessive inborn error of metabolism caused by the deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolism pathway. This defect results in accumulation of succinylacetone (SA) that reacts with amino acids and proteins to form stable adducts via Schiff base formation, lysine being the most reactive amino acid. HT1 patients surviving beyond infancy are at considerable risk for the development of hepatocellular carcinoma, and a high level of chromosomal breakage is observed in HT1 cells, suggesting a defect in the processing of DNA. In this paper we show that the overall DNA-ligase activity is low in HT1 cells (about 20% of the normal value) and that Okazaki fragments are rejoined at a reduced rate compared with normal fibroblasts. No mutation was found by sequencing the ligase I cDNA from HT1 cells, and the level of expression of the ligase I mRNA was similar in normal and HT1 fibroblasts, suggesting the presence of a ligase inhibitor. SA was shown to inhibit in vitro the overall DNA-ligase activity present in normal cell extracts. The activity of purified T4 DNA-ligase, whose active site is also a lysine residue, was inhibited by SA in a dose-dependent manner. These results suggest that accumulation of SA reduces the overall ligase activity in HT1 cells and indicate that metabolism errors may play a role in regulating enzymatic activities involved in DNA replication and repair.

Delayed loss of cholesterol from a localized lipoprotein depot in apolipoprotein A-I-deficient mice

The anti-atherogenic role of high density lipoprotein is well known even though the mechanism has not been established. In this study, we have used a novel model system to test whether removal of lipoprotein cholesterol from a localized depot will be affected by apolipoprotein A-I (apo A-I) deficiency. We compared the egress of cholesterol injected in the form of cationized low density lipoprotein into the rectus femoris muscle of apo A-I K-O and control mice. When the injected lipoprotein had been labeled with [3H]cholesterol, the t½ of labeled cholesterol loss from the muscle was about 4 days in controls and more than 7 days in apo A-I K-O mice. The loss of cholesterol mass had an initial slow (about 4 days) and a later more rapid component; after day 4, the disappearance curves for apo A-I K-O and controls began to diverge, and by day 7, the loss of injected cholesterol was significantly slower in apo A-I K-O than in controls. The injected lipoprotein cholesterol is about 70% in esterified form and undergoes hydrolysis, which by day 4 was similar in control and apo A-I K-O mice. The efflux potential of serum from control and apo A-I K-O mice was studied using media containing 2% native or delipidated serum. A significantly lower efflux of [3H]cholesterol from macrophages was found with native and delipidated serum from apo A-I K-O mice. In conclusion, these findings show that lack of apo A-I results in a delay in cholesterol loss from a localized depot in vivo and from macrophages in culture. These results provide support for the thesis that anti-atherogenicity of high density lipoprotein is related in part to its role in cholesterol removal.

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein

A challenge for subunit vaccines whose goal is to elicit CD8+ cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.

Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanism

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

“Switch I” mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by σ54-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of σ54-holoenzyme—residues 206–220—is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrCA216V and NtrCG219K, have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrCA216C and NtrCG219C, are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix–turn–helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with σ54-holoenzyme. The conserved region in which these amino acid substitutions occur (206–220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21ras.

An interaction between DNA ligase I and proliferating cell nuclear antigen: Implications for Okazaki fragment synthesis and joining

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol δ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol δ. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol δ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol δ, disrupts the DNA ligase I–PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA–DNA pol δ complex, DNA pol δ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.

The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA

tRNA pseudouridine synthase I (ΨSI) catalyzes the conversion of uridine to Ψ at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. ΨSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNAPhe containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and ΨSI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for ΨS.

The emergence of form by replication

It is shown with a simple mathematical model that if a system exhibits a given form (a spatial structure) and is put in contact with another system of the same type but in a state of spatial disorder, then under certain conditions their mutual interaction as they evolve in time allows replication of form in the disordered system with a controllable degree of faithfulness.