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A K Aggarwal

Publications and source records attributed to A K Aggarwal.

At least 37 records · Page 2Linked to original sources
PubMed2000

Crystallization and characterization of Pumilo: a novel RNA binding protein.

Axis determination in early Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6(3) with unit cell dimensions of a = b = 94.5 A, c = 228.9 A, alpha = beta = 90 degrees, gamma = 120 degrees and diffract to 2.6 A with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of alpha-helical secondary structure.

Animals↗
PubMed2000

Adolescent behaviour regarding reproductive health.

A cross-sectional household survey was undertaken in rural areas of district Sirmaur, Himachal Pradesh, to assess the knowledge, beliefs and practices of adolescents about reproductive health. Six hundred and forty three unmarried adolescents aged 15-19 years were selected by 30-cluster sampling method from 2400 households. They were interviewed using a semi-structured schedule. Fifty-six per cent were girls. More girls (14%) were illiterate than boys (6%). Most of the boys (88%) and 58% girls knew that a female conceives through sexual intercourse. Seventy seven per cent girls and eighty seven per cent boys were aware of at least one contraceptive method. Majority of the girls (71%) and boys (82.5%) favoured termination of an unwanted pregnancy. About one-fourth respondents considered husband responsible for infertility and for sex of the baby. Boys considered night emission, poor body built and less growth of hair as reproductive health problems, whereas, girls were worried about menstruation and inadequate breast development. Almost 6% boys reported use of a contraceptive method indicating existence of pre-marital sexual activity. Knowledge on reproductive health is low and there is a big gap between actual and desired practices.

Adolescent↗
PubMed2000

Structure of BamHI bound to nonspecific DNA: a model for DNA sliding.

The central problem faced by DNA binding proteins is how to select the correct DNA sequence from the sea of nonspecific sequences in a cell. The problem is particularly acute for bacterial restriction enzymes because cleavage at an incorrect DNA site could be lethal. To understand the basis of this selectivity, we report here the crystal structure of endonuclease BamHI bound to noncognate DNA. We show that, despite only a single base pair change in the recognition sequence, the enzyme adopts an open configuration that is on the pathway between free and specifically bound forms of the enzyme. Surprisingly, the DNA drops out of the binding cleft with a total loss of base-specific and backbone contacts. Taken together, the structure provides a remarkable snapshot of an enzyme poised for linear diffusion (rather than cleavage) along the DNA.

Crystallography, X-Ray↗
PubMed2000

Gene repression by coactivator repulsion.

We show that the IRF-2 oncoprotein represses virus-induced IFN-beta gene transcription via a novel mechanism. Virus infection induces recruitment of IRF-2 to some of the endogenous IFN-beta enhancers as part of the enhanceosome. Enhanceosomes bearing IRF-2 cannot activate transcription, due to the presence of a domain in IRF-2 that prevents enhanceosome-dependent recruitment of the CBP-Pol II holoenzyme complex. As a consequence, IRF-2 incorporation into enhanceosomes restricts the number of IFN-beta promoters directing transcription. Remarkably, deletion of the IRF-2 gene increases IFN-beta expression by expanding the number of cells capable of inducing IFN-beta gene transcription in response to virus infection.

Animals↗
PubMed2000

Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution.

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1. 5 A resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar alpha/beta core, but in BglII, the core is augmented by a beta-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein-DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.

Bacterial Proteins↗
PubMed2000

Otomycosis: a clinicomycologic study.

Otomycosis is a common fungal infection of the ear that is seen in the tropical and subtropical regions of the world. We performed mycologic analyses on debris and scraping samples from the external ear canals of 95 patients who had been clinically diagnosed with otomycosis. Seventy-one samples (74.7%) were positive for fungal growth; two of these samples contained two fungi, bringing the total number of isolates to 73. The most common pathogens were Aspergillus fumigatus (41.1% of all isolates), A niger (36.9%), and Candida albicans (8.2%).

Adult↗
PubMed1999

Structure and ligand of a histone acetyltransferase bromodomain.

Histone acetylation is important in chromatin remodelling and gene activation. Nearly all known histone-acetyltransferase (HAT)-associated transcriptional co-activators contain bromodomains, which are approximately 110-amino-acid modules found in many chromatin-associated proteins. Despite the wide occurrence of these bromodomains, their three-dimensional structure and binding partners remain unknown. Here we report the solution structure of the bromodomain of the HAT co-activator P/CAF (p300/CBP-associated factor). The structure reveals an unusual left-handed up-and-down four-helix bundle. In addition, we show by a combination of structural and site-directed mutagenesis studies that bromodomains can interact specifically with acetylated lysine, making them the first known protein modules to do so. The nature of the recognition of acetyl-lysine by the P/CAF bromodomain is similar to that of acetyl-CoA by histone acetyltransferase. Thus, the bromodomain is functionally linked to the HAT activity of co-activators in the regulation of gene transcription.

Acetyltransferases↗
PubMed1999

Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types.

The mechanisms by which transient gradients of signaling molecules lead to emergence of specific cell types remain a central question in mammalian organogenesis. Here, we demonstrate that the appearance of four ventral pituitary cell types is mediated via the reciprocal interactions of two transcription factors, Pit1 and GATA2, which are epistatic to the remainder of the cell type-specific transcription programs and serve as the molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA binding-independent function of Pit1, suppressing the ventral GATA2-dependent gonadotrope program by inhibiting GATA2 binding to gonadotrope- but not thyrotrope-specific genes, indicating that both DNA binding-dependent and -independent actions of abundant determining factors contribute to generate distinct cell phenotypes.

Amino Acid Sequence↗
PubMed1999

Structure of a DNA-bound Ultrabithorax-Extradenticle homeodomain complex.

During the development of multicellular organisms, gene expression must be tightly regulated, both spatially and temporally. One set of transcription factors that are important in animal development is encoded by the homeotic (Hox) genes, which govern the choice between alternative developmental pathways along the anterior-posterior axis. Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbxl in mammals). To understand the structural basis of Hox-Extradenticle pairing, we determine here the crystal structure of an Ultrabithorax-Extradenticle-DNA complex at 2.4 A resolution, using the minimal polypeptides that form a cooperative heterodimer. The Ultrabithorax and Extradenticle homeodomains bind opposite faces of the DNA, with their DNA-recognition helices almost touching each other. However, most of the cooperative interactions arise from the YPWM amino-acid motif of Ultrabithorax-located amino-terminally to its homeodomain-which forms a reverse turn and inserts into a hydrophobic pocket on the Extradenticle homeodomain surface. Together, these protein-DNA and protein-protein interactions define the general principles by which homeotic proteins interact with Extradenticle (or Pbx1) to affect development along the anterior-posterior axis of animals.

Amino Acid Sequence↗
PubMed1999

Community based study of reproductive tract infections among ever married women of reproductive age in a rural area of Haryana, India.

A community based study was carried out to estimate the load of reproductive tract infections (RTIs) among ever married rural women aged 15-44 years and utilisation of a mobile village based treatment facility by them, during 1997. Complete household survey was done in 10 randomly selected villages of Mahindergarh district in Haryana, India. A total of 2325 women were interviewed by auxiliary nurse midwives and were invited for medical examination in a health camp set-up in their village. Sixty-one percent (1415/2325) women reported symptoms of RTIs. Only 35% (812/2325) had their per-speculum examination done. Out of those examined, 32% (263/812) had vaginitis, 21% (175/812) had cervicits, and 19% (156/812) had pelvic inflammatory disease. Vaginal smear of those having discharge revealed that 48% (231/476) had bacterial vaginosis, 0.8% (4/476) had fungal infection 9% (44/496) had trichomonal infection and none was found to be having gonococcal infection. Tests for chlamydial infection could not be performed as the cost was not affordable. Eighty-nine percent of women who reported symptoms of RTIs had not consulted anyone for their problem prior to our village based camp. Only 42% symptomatic and 24% asymptomatics availed the services even in the village based camps. Study revealed high load of reproductive tract infections and low utilisation of treatment facility. In-depth studies are required to understand for high RTI morbidity load and low treatment seeking rate so as to design an appropriate RTI control programme.

Adolescent↗
PubMed1998

Signal-specific co-activator domain requirements for Pit-1 activation.

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes. Here we show that whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT, mSin3A/B and histone deacetylases, and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, the histone acetyltransferase functions of CBP or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors.

Acetyltransferases↗
PubMed1998

Structure of FokI has implications for DNA cleavage.

FokI is a member an unusual class of restriction enzymes that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence. FokI consists of an N-terminal DNA recognition domain and a C-terminal cleavage domain. The bipartite nature of FokI has led to the development of artificial enzymes with novel specificities. We have solved the structure of FokI to 2.3 A resolution. The structure reveals a dimer, in which the dimerization interface is mediated by the cleavage domain. Each monomer has an overall conformation similar to that found in the FokI-DNA complex, with the cleavage domain packing alongside the DNA recognition domain. In corroboration with the cleavage data presented in the accompanying paper in this issue of Proceedings, we propose a model for FokI DNA cleavage that requires the dimerization of FokI on DNA to cleave both DNA strands.

Amino Acid Sequence↗
PubMed1998

FokI dimerization is required for DNA cleavage.

FokI is a type IIs restriction endonuclease comprised of a DNA recognition domain and a catalytic domain. The structural similarity of the FokI catalytic domain to the type II restriction endonuclease BamHI monomer suggested that the FokI catalytic domains may dimerize. In addition, the FokI structure, presented in an accompanying paper in this issue of Proceedings, reveals a dimerization interface between catalytic domains. We provide evidence here that FokI catalytic domain must dimerize for DNA cleavage to occur. First, we show that the rate of DNA cleavage catalyzed by various concentrations of FokI are not directly proportional to the protein concentration, suggesting a cooperative effect for DNA cleavage. Second, we constructed a FokI variant, FokN13Y, which is unable to bind the FokI recognition sequence but when mixed with wild-type FokI increases the rate of DNA cleavage. Additionally, the FokI catalytic domain that lacks the DNA binding domain was shown to increase the rate of wild-type FokI cleavage of DNA. We also constructed an FokI variant, FokD483A, R487A, which should be defective for dimerization because the altered residues reside at the putative dimerization interface. Consistent with the FokI dimerization model, the variant FokD483A, R487A revealed greatly impaired DNA cleavage. Based on our work and previous reports, we discuss a pathway of DNA binding, dimerization, and cleavage by FokI endonuclease.

Base Sequence↗
PubMed1998

Structure of IRF-1 with bound DNA reveals determinants of interferon regulation.

The family of interferon regulatory factor (IRF) transcription factors is important in the regulation of interferons in response to infection by virus and in the regulation of interferon-inducible genes. The IRF family is characterized by a unique 'tryptophan cluster' DNA-binding region. Here we report the crystal structure of the IRF-1 region bound to the natural positive regulatory domain I (PRD I) DNA element from the interferon-beta promoter. The structure provides the first three-dimensional view of a member of the growing IRF family, revealing a new helix-turn-helix motif that latches onto DNA through three of the five conserved tryptophans. The motif selects a short GAAA core sequence through an obliquely angled recognition helix, with an accompanying bending of the DNA axis in the direction of the protein. Together, these features suggest a basis for the occurrence of GAAA repeats within IRF response elements and provide clues to the assembly of the higher-order interferon-beta enhancesome.

Amino Acid Sequence↗
PubMed1998

Novel site-specific DNA endonucleases.

Site-specific hydrolysis of DNA is common to many biological processes. Three new structures, FokI, I-CreI and PI-SceI, were reported in the past year, providing the first view of type IIs endonucleases and homing endonucleases. Together, they reveal an extraordinary set of new mechanisms by which endonucleases target the hydrolysis of specific DNA sequences.

Amino Acid Sequence↗
PubMed1998

The role of metals in catalysis by the restriction endonuclease BamHI.

Type II restriction enzymes are characterized by their remarkable specificity and simplicity. They require only divalent metals (such as Mg2+ or Mn2+) as cofactors to catalyze the hydrolysis of DNA. However, most of the structural work on endonucleases has been performed in the absence of metals, leaving unanswered questions about their mechanisms of DNA cleavage. Here we report structures of the endonuclease BamHI-DNA complex, determined in the presence of Mn2+ and Ca2+, that describe the enzyme at different stages of catalysis. Overall, the results support a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of EcoRV.

Binding Sites↗