Identification and partial purification of a protein binding to the human immunoglobulin kappa enhancer kappa E2 site.

We previously described a domain in the 5'' half of the human immunoglobulin kappa enhancer which could bind nuclear proteins in vitro, as detected by a lambda exonuclease protection assay. A second more 3'' binding domain in the enhancer has now been detected by a similar assay employing a different exonuclease, the T7 gene 6 exonuclease. Using this assay and starting with a pig spleen nuclear extract, we have purified 5000-fold a protein that binds to the 3'' domain. In a DNase I footprint experiment the partially purified protein protects a 27 bp segment in the enhancer centered around the sequence CAGGTGGC, which corresponds to the kappa E2 sequence motif described in the mouse kappa enhancer. The protein, designated NF-kappa E2, also appears to bind at a position downstream of kappa E2, at or near the kappa E3 site. Proteins capable of binding at kappa E2 are found in several mammalian species and are expressed in both lymphoid and non-lymphoid tissues.     

A duplicated HNF-3 binding site in the CYP2H2 promoter underlies the weak phenobarbital induction response

We are investigating induction of chicken cytochrome P450 genes by the sedative phenobarbital in chick embryo hepatocytes. The steady-state level of induced mRNA for the gene CYP2H1 is about 10-fold higher than that of a second gene, CYP2H2. Here, we show that a difference in drug-responsive enhancer activity does not underlie the differential response of these genes to phenobarbital since upstream enhancer regions are identical in these genes. The first 198 bp of CYP2H2 promoter sequence is identical to the CYP2H1 gene promoter, except that the functional HNF-3 binding site in the CYP2H1 promoter is replaced with a duplicated HNF-3 sequence in the CYP2H2 promoter. Transient expression analysis established that the promoter activity of the CYP2H2 gene was about ninefold lower than the CYP2H1 gene. Mutagenesis of either of the partially overlapping HNF-3 sites in the CYP2H2 gene substantially induced drug induction. Gel-shift analysis established that each of these HNF-3 sites bound HNF-3, most likely HNF-3beta. In-vitro footprint analysis demonstrated that all the identified sites in the CYP2H2 promoter bound protein except the duplicated HNF-3 region. However, protein binding was observed by in-vitro footprint analysis if either of the HNF-3 sites was mutated in the CYP2H2 promoter. Hence, duplication of the HNF-3 site in the CYP2H2 promoter does not allow binding of HNF-3 in the promoter context and may be predominantly, if not exclusively, responsible for the poor response of the CYP2H2 gene to phenobarbital.     

Systematic analysis of sequence variability of the endothelin-1 gene: a prerequisite for association studies

We analyzed allele frequencies and pairwise linkage disequilibria of 13 variants in the EDN1 gene of 298 young males, the majority of German ancestry. Our analysis comprises all common variants in the five exons and flanking intronic regions, as well as known polymorphisms in the promoter sequence. In addition to previously analyzed polymorphisms, our haplotype reconstruction included five recently described variants and was done by using three different algorithms to allow inference of result stability. More than 30 haplotypes were predicted. All haplotypes with frequencies > or = 1% were inferred by all three methods and can be described by seven haplotype tagging single-nucleotide polymorphisms (htSNPs), reducing the genotyping load to 65%. Three of these haplotypes with frequencies of about 11%, 9%, and 4% had been mistaken for one haplotype in the previous analysis, which included only six polymorphisms, some of them not being htSNPs. Systematic analysis of sequence variability and comprehensive haplotype analysis of the EDN1 gene determined a substantial part of its genetic variability for further association studies and helped to reduce the genotyping load for common phenotypes.     

A locus-wide approach to assessing variation in the avian MHC: the B-locus of the wild turkey

Studies of major histocompatibility complex (MHC) diversity in non-model vertebrates typically focus on structure and sequence variation in the antigen-presenting loci: the highly variable and polymorphic class I and class IIB genes. Although these studies provide estimates of the number of genes and alleles/locus, they often overlook variation in functionally related and co-inherited genes important in the immune response. This study utilizes the sequence of the MHC B-locus derived from a commercial turkey to investigate MHC variation in wild birds. Sequences were obtained for nine interspersed MHC amplicons (non-class I/II) from each of 40 birds representing 3 subspecies of wild turkey (Meleagris gallopavo). Analysis of aligned sequences identified 238 single-nucleotide variants approximately one-third of which had minor allele frequencies >0.2 in the sampled birds. PHASE analysis identified 70 prospective MHC haplotypes in the wild turkeys, whereas a combined analysis with commercial birds identified almost 100 haplotypes in the species. Denaturing gradient gel electrophoresis (DGGE) of the class IIB loci was used to test the efficacy of single-nucleotide polymorphism (SNP) haplotyping to capture locus-wide variation. Diversity in SNP haplotypes and haplotype sharing among individuals was directly reflected in the DGGE patterns. Utilization of a reference haplotype to sequence interspersed regions of the MHC has significant advantages over other methods of surveying diversity while identifying high-frequency SNPs for genotyping. SNP haplotyping provides a means to identify both divergent haplotypes and homozygous individuals for assessment of immunological variation in wild and domestic populations.     

Development of a novel mammalian cell surface antibody display platform

Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically use prokaryotic organisms such as phage and bacteria or lower eukaryotic organisms, such as yeast. These organisms possess either no or different post-translational modification functions from mammalian cells and prefer to display small antibody fragments instead of full-length IgGs. We report here a novel mammalian cell-based antibody display platform that displays full-length functional antibodies on the surface of mammalian cells. Through recombinase-mediated DNA integration, each host cell contains one copy of the gene of interest in the genome. Utilizing a hot-spot integration site, the expression levels of the gene of interest are high and comparable between clones, ensuring a high signal to noise ratio. Coupled with fluorescence-activated cell sorting (FACS) technology, our platform is high throughput and can distinguish antibodies with very high antigen binding affinities directly on the cell surface. Single-round FACS can enrich high affinity antibodies by more than 500-fold. Antibodies with significantly improved neutralizing activity have been identified from a randomly mutagenized library, demonstrating the power of this platform in screening and selecting antibody therapeutics.Key words: antibody display, mammalian display, antibody library, vector, antibody screen, affinity maturation     

Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites

Deletion analysis of the human PRL promoter in endometrial stromal cells decidualized in vitro revealed a 536-bp enhancer located between nucleotide (nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragment ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cells. DNase I footprint analysis of decidualized endometrial stromal cells revealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind activator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 complex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 536-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer activity by approximately 70%. Conversely, coexpression of Fra-2 in combination with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. Introduction of JunD and Fra-2 into nondecidual cells is sufficient to confer enhancer activity. JunD and Fra-2 protein expression was markedly increased in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These investigations indicate that the 5'-flanking region of the human PRL gene contains a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding sites within this enhancer region are critical for activity.     

Reference genome sequence of the model plant Setaria

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ～400-Mb assembly covers ～80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species that demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).     

Experimental Resurrection of Ancestral Mammalian CPEB3 Ribozymes Reveals Deep Functional Conservation

Self-cleaving ribozymes are genetic elements found in all domains of life, but their evolution remains poorly understood. A ribozyme located in the second intron of the cytoplasmic polyadenylation binding protein 3 gene (CPEB3) shows high sequence conservation in mammals, but little is known about the functional conservation of self-cleaving ribozyme activity across the mammalian tree of life or during the course of mammalian evolution. Here, we use a phylogenetic approach to design a mutational library and a deep sequencing assay to evaluate the in vitro self-cleavage activity of numerous extant and resurrected CPEB3 ribozymes that span over 100 My of mammalian evolution. We found that the predicted sequence at the divergence of placentals and marsupials is highly active, and this activity has been conserved in most lineages. A reduction in ribozyme activity appears to have occurred multiple different times throughout the mammalian tree of life. The in vitro activity data allow an evaluation of the predicted mutational pathways leading to extant ribozyme as well as the mutational landscape surrounding these ribozymes. The results demonstrate that in addition to sequence conservation, the self-cleavage activity of the CPEB3 ribozyme has persisted over millions of years of mammalian evolution.