Human SM22α BAC encompasses regulatory sequences for expression in vascular and visceral smooth muscles at fetal and adult stages

2003 ◽  
Vol 284 (4) ◽  
pp. H1398-H1407 ◽  
Author(s):  
Rui Xu ◽  
Ye-Shih Ho ◽  
Raquel P. Ritchie ◽  
Li Li
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Dna Sequences ◽  
Expression Patterns ◽  
Smooth Muscles ◽  
Artificial Chromosome ◽  
Developmental Expression ◽  
Regulatory Mechanisms ◽  
Regulatory Sequences ◽  
Cardiovascular Development ◽  
Evolutionarily Conserved

The SM22α gene has widely been used to study the regulatory mechanisms of smooth muscle cell (SMC) gene expression during cardiovascular development. To determine the regulatory mechanisms for the evolutionarily conserved human SM22α (h SM22α) gene, we demonstrated that 445 bp upstream DNA sequences of h SM22α gene exhibited a high transcriptional activity in arterial SMC, not in venous nor in visceral SMCs during embryogensis. However, this promoter was gradually turned off in adulthood. Inclusion of the first intron in this promoter suppressed the promoter activity in pulmonary trunk arterial SMCs, whereas the expression in other systemic vasculature remained similar to that of the h SM22-445 promoter during the fetal and adult stages. To determine whether additional sequences are required for SM22α expression in all subtypes of SMCs, we examined the expression of a bacterial artificial chromosome containing the h SM22α locus in transgenic mice. The h SM22α transgene showed similar developmental expression patterns as the endogenous mouse SM22α gene, suggesting that this bacterial artificial chromosome contains essential regulatory sequences for its expression in arterial, venous, and visceral tissues during development.

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2)

Genome ◽  
2001 ◽  
Vol 44 (2) ◽  
pp. 154-162 ◽  
Author(s):  
Meizhong Luo ◽  
Yi-Hong Wang ◽  
David Frisch ◽  
Tarek Joobeur ◽  
Rod A Wing ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Fusarium Wilt ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Microtiter Plates ◽  
Bac Libraries ◽  
Average Size ◽  
Improved Methods

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.Key words: bacterial artificial chromosome (BAC) library, Fusarium wilt, melon, pCUGIBAC1, resistant gene.

Transgenic modification of cows milk for value-added processing

1998 ◽  
Vol 10 (8) ◽  
pp. 671 ◽  
Author(s):  
Kurt A. Zuelke
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Dairy Cattle ◽  
Dna Sequences ◽  
Gene Locus ◽  
Bac Library ◽  
Value Added ◽  
Artificial Chromosome ◽  
Coordinated Expression ◽  
Casein Genes ◽  
Transgenic Technologies

The application of transgenic technologies in dairy cattle has been restricted largely to producing potential pharmaceutical or nutriceutical products in the mammary gland. Broader application of transgenesis in dairy cattle production will require identifying target traits that are both amenable to transgenic modification and economically important to the dairy industry. The casein proteins are the most valuable component of cows milk destined for value-added processing. The four bovine casein genes lie within a single, multi-gene locus of approximately 200 kb in length. The working hypothesis is that this multi-gene locus contains all of the DNA sequences required to regulate the coordinated expression of all four individual casein genes (i.e. a locus control region or LCR). The initial research aim is to clone the entire casein locus into a bacterial artificial chromosome (BAC) vector, thus preserving the extended 5′and 3′ regions that flank the locus, as well as maintaining the spatial integrity of the four individual casein genes that comprise the locus. The author's laboratory has prepared a bacterial artificial chromosome (BAC) library of genomic DNA from elite dairy cattle. Partial, non-elite BAC clones of the casein gene locus are being tested in transgenic mice to establish proof of concept. Advances in nuclear transfer of transfected somatic cells should improve the efficiency of producing transgenic calves that possess a BAC casein construct introduced into an elite genetic background.

scholarly journals Retroviral Transcriptional Regulation and Embryonic Stem Cells: War and Peace

2014 ◽  
Vol 35 (5) ◽  
pp. 770-777 ◽  
Author(s):  
Sharon Schlesinger ◽  
Stephen P. Goff
Keyword(s):  
Dna Sequences ◽  
Regulatory Networks ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Cell Types ◽  
Endogenous Retroviruses ◽  
Regulatory Sequences ◽  
Cellular Genes ◽  
Wide Range

Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps “noisy” control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks.

scholarly journals Bmp2 Transcription in Osteoblast Progenitors Is Regulated by a Distant 3′ Enhancer Located 156.3 Kilobases from the Promoter

2007 ◽  
Vol 27 (8) ◽  
pp. 2934-2951 ◽  
Author(s):  
Ronald L. Chandler ◽  
Kelly J. Chandler ◽  
Karen A. McFarland ◽  
Douglas P. Mortlock
Keyword(s):  
Risk Factor ◽  
Osteoblast Differentiation ◽  
Expression Patterns ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
Bone Morphogenetic Protein 2 ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Bac Clone ◽  
Osteoblast Progenitor

ABSTRACT Bone morphogenetic protein 2 (encoded by Bmp2) has been implicated as an important signaling ligand for osteoblast differentiation and bone formation and as a genetic risk factor for osteoporosis. To initially survey a large genomic region flanking the mouse Bmp2 gene for cis-regulatory function, two bacterial artificial chromosome (BAC) clones that extend far upstream and downstream of the gene were engineered to contain a lacZ reporter cassette and tested in transgenic mice. Each BAC clone directs a distinct subset of normal Bmp2 expression patterns, suggesting a modular arrangement of distant Bmp2 regulatory elements. Strikingly, regulatory sequences required for Bmp2 expression in differentiating osteoblasts, as well as tooth buds, hair placodes, kidney, and other tissues, are located more than 53 kilobases 3′ to the promoter. By testing BACs with engineered deletions across this distant 3′ region, we parsed these regulatory elements into separate locations and more closely refined the location of the osteoblast progenitor element. Finally, a conserved osteoblast progenitor enhancer was identified within a 656-bp sequence located 156.3 kilobases 3′ from the promoter. The identification of this enhancer should permit further investigation of upstream regulatory mechanisms that control Bmp2 transcription during osteoblast differentiation and are relevant to further studies of Bmp2 as a candidate risk factor gene for osteoporosis.

scholarly journals Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Alberto Stolfi ◽  
Elijah K Lowe ◽  
Claudia Racioppi ◽  
Filomena Ristoratore ◽  
C Titus Brown ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Embryonic Cell ◽  
Regulatory Mechanisms ◽  
Regulatory Sequences ◽  
Pharyngeal Muscle ◽  
Fate Specification ◽  
Cell Lineages ◽  
Identical Cell ◽  
Cis And Trans

Ascidians present a striking dichotomy between conserved phenotypes and divergent genomes: embryonic cell lineages and gene expression patterns are conserved between distantly related species. Much research has focused on Ciona or Halocynthia spp. but development in other ascidians remains poorly characterized. In this study, we surveyed the multipotent myogenic B7.5 lineage in Molgula spp. Comparisons to the homologous lineage in Ciona revealed identical cell division and fate specification events that result in segregation of larval, cardiac, and pharyngeal muscle progenitors. Moreover, the expression patterns of key regulators are conserved, but cross-species transgenic assays uncovered incompatibility, or ‘unintelligibility’, of orthologous cis-regulatory sequences between Molgula and Ciona. These sequences drive identical expression patterns that are not recapitulated in cross-species assays. We show that this unintelligibility is likely due to changes in both cis- and trans-acting elements, hinting at widespread and frequent turnover of regulatory mechanisms underlying otherwise conserved aspects of ascidian embryogenesis.

Screening regulatory sequences from bacterial artificial chromosome DNA of alpha- and beta-globin gene clusters

2002 ◽  
Vol 80 (4) ◽  
pp. 415-420 ◽  
Author(s):  
Shen Zhang ◽  
Hua-Bing Zhang ◽  
De-Pei Liu ◽  
Xing-Guo Li ◽  
De-Long Hao ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Globin Gene ◽  
K562 Cells ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Nuclear Proteins ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Regulatory Dna

In the forthcoming postgenomic era, identification of regulatory DNA sequences is becoming increasingly important for characterizing DNA-binding proteins and for elucidating the regulatory mechanisms of gene expression. Presently, there lack efficient methods to broadly screen and identify DNA regulatory elements on a large scale. We established herein an efficient strategy to screen regulatory sequences from bacterial artificial chromosome (BAC) DNAs containing human alpha- and beta-globin gene clusters based on polymerase chain reaction and electrophoretic mobility shift assay (EMSA) techniques without purified transcription factors. Twenty-three subclones derived from alpha-BAC DNA by bulk EMSA selection retained the ability to bind nuclear proteins of K562 cells when retested by EMSA. In 19 clones sequenced, 14 are identical to those registered in GenBank and five have one base difference. All of the 24 randomly picked beta-BAC clones showed specific binding with nuclear proteins of K562 cells. In 11 clones sequenced, eight are identical to those registered in GenBank and three have one base difference. This approach could be particularly powerful if combined with other systematic methods for identifying cis-regulatory DNA elements.Key words: gene expression regulation, bacterial artificial chromosome, alpha- and beta-globin gene clusters.

scholarly journals GimmeMotifs: an analysis framework for transcription factor motif analysis

2018 ◽  
Author(s):  
Niklas Bruse ◽  
Simon J. van Heeringen
Keyword(s):  
Dna Sequences ◽  
Motif Discovery ◽  
High Throughput Sequencing ◽  
Performance Metrics ◽  
De Novo ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Ensemble Method ◽  
Regulatory Sequences ◽  
Motif Analysis

AbstractBackgroundTranscription factors (TFs) bind to specific DNA sequences, TF motifs, in cis-regulatory sequences and control the expression of the diverse transcriptional programs encoded in the genome. The concerted action of TFs within the chromatin context enables precise temporal and spatial expression patterns. To understand how TFs control gene expression it is essential to model TF binding. TF motif information can help to interpret the exact role of individual regulatory elements, for instance to predict the functional impact of non-coding variants.FindingsHere we present GimmeMotifs, a comprehensive computational framework for TF motif analysis. Compared to the previously published version, this release adds a whole range of new functionality and analysis methods. It now includes tools for de novo motif discovery, motif scanning and sequence analysis, motif clustering, calculation of performance metrics and visualization. Included with GimmeMotifs is a non-redundant database of clustered motifs. Compared to other motif databases, this collection of motifs shows competitive performance in discriminating bound from unbound sequences. Using our de novo motif discovery pipeline we find large differences in performance between de novo motif finders on ChIP-seq data. Using an ensemble method such as implemented in GimmeMotifs will generally result in improved motif identification compared to a single motif finder. Finally, we demonstrate maelstrom, a new ensemble method that enables comparative analysis of TF motifs between multiple high-throughput sequencing experiments, such as ChIP-seq or ATAC-seq. Using a collection of ~200 H3K27ac ChIP-seq data sets we identify TFs that play a role in hematopoietic differentiation and lineage commitment.ConclusionGimmeMotifs is a fully-featured and flexible framework for TF motif analysis. It contains both command-line tools as well as a Python API and is freely available at: https://github.com/vanheeringen-lab/gimmemotifs.

scholarly journals Modeling Enhancer-Promoter Interactions with Attention-Based Neural Networks

2017 ◽  
Author(s):  
Weiguang Mao ◽  
Dennis Kostka ◽  
Maria Chikina
Keyword(s):  
Dna Sequences ◽  
Target Genes ◽  
Expression Patterns ◽  
Regulatory Sequences ◽  
Learning Approaches ◽  
Interaction Prediction ◽  
Genome Wide ◽  
Gene Regulatory ◽  
Enhancer Function ◽  
Enhancer Sequences

AbstractBackgroundGene regulatory sequences play critical roles in ensuring tightly controlled RNA expression patterns that are essential in a large variety of biological processes. Specifically, enhancer sequences drive expression of their target genes, and the availability of genome-wide maps of enhancer-promoter interactions has opened up the possibility to use machine learning approaches to extract and interpret features that define these interactions in different biological contexts.MethodsInspired by machine translation models we develop an attention-based neural network model, EPIANN, to predict enhancer-promoter interactions based on DNA sequences. Codes and data are available at https://github.com/wgmao/EPIANN.ResultsOur approach accurately predicts enhancer-promoter interactions across six cell lines. In addition, our method generates pairwise attention scores at the sequence level, which specify how short regions in the enhancer and promoter pair-up to drive the interaction prediction. This allows us to identify over-represented transcription factors (TF) binding sites and TF-pair interactions in the context of enhancer function.

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