scholarly journals Multiple, Distant Gata2 Enhancers Specify Temporally and Tissue-Specific Patterning in the Developing Urogenital System

2004 ◽  
Vol 24 (23) ◽  
pp. 10263-10276 ◽  
Author(s):  
Melin Khandekar ◽  
Norio Suzuki ◽  
Jon Lewton ◽  
Masayuki Yamamoto ◽  
James Douglas Engel
Keyword(s):  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Specific Pattern ◽  
Regulatory Sequence ◽  
General Strategy ◽  
Urogenital System ◽  
Loss Of Function ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
Null Mutant Mice

ABSTRACT Transcription factor GATA-2 is expressed in a complex temporally and tissue-specific pattern within the developing embryo. Loss-of-function studies in the mouse showed that GATA-2 activity is first required during very early hematopoiesis. We subsequently showed that a 271-kbp yeast artificial chromosome (YAC) transgene could fully complement the loss of Gata2 hematopoietic function but that these YAC-rescued Gata2 null mutant mice die perinatally due to defective urogenital development. The rescuing YAC did not display appropriate urogenital expression of Gata2, implying the existence of a urogenital-specific enhancer(s) lying outside the boundaries of this transgene. Here we outline a coupled general strategy for regulatory sequence discovery, linking bioinformatics to functional genomics based on the bacterial artificial chromosome (BAC) libraries used to generate the mouse genome sequence. Exploiting this strategy, we screened >1 Mbp of genomic DNA surrounding Gata2 for urogenital enhancer activity. We found that the spatially and tissue-specific functions for Gata2 in the developing urogenital system are conferred by at least three separate regionally and temporally specific urogenital enhancer elements, two of which reside far 3′ to the Gata2 structural gene. Including the additional enhancers that were discovered using this strategy (called BAC trap) extends the functional realm of the Gata2 locus to greater than 1 Mbp.

scholarly journals Identification of an enhancer required for the expression of a mouse major urinary protein gene in the submaxillary gland.

1991 ◽  
Vol 11 (8) ◽  
pp. 4244-4252 ◽  
Author(s):  
H J Son ◽  
K Shahan ◽  
M Rodriguez ◽  
E Derman ◽  
F Costantini
Keyword(s):  
Transcription Initiation ◽  
Fusion Gene ◽  
Submaxillary Gland ◽  
Regulatory Elements ◽  
Specific Pattern ◽  
Regulatory Sequence ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
Dna Fragment

The MUP1.5b gene was previously found to be expressed specifically in the submaxillary gland and at high levels when introduced into mice as a transgene including 4.7 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. To localize regulatory elements responsible for this tissue-specific pattern of expression, we tested the expression of three additional MUP1.5b transgenes including various amounts of 5'-flanking DNA. These experiments indicated that sequences between -1.85 and -3.46 kb from the transcription initiation site were required for high-level expression in the submaxillary gland. The presence of regulatory elements in this region was also suggested by the detection of a DNase I-hypersensitive site, seen only in submaxillary gland nuclei, at position -2.5 kb upstream from the MUP1.5a gene, a member of the same MUP gene subfamily and virtually identical to the MUP1.5b gene. Further evidence for enhancer activity was provided by the ability of the 1.6-kb DNA fragment including sequences between -1.85 and -3.46 kb to stimulate the expression of an otherwise inactive MUP1.5b-chloramphenicol acetyltransferase fusion gene specifically in the submaxillary gland. The nucleotide sequence of this 1.6-kb DNA fragment was found to be identical for the MUP1.5a and MUP1.5b genes. Together, these results provide the first localization of a cis-acting regulatory sequence involved in the differential tissue-specific expression of the MUP gene family.

Identification of an enhancer required for the expression of a mouse major urinary protein gene in the submaxillary gland

1991 ◽  
Vol 11 (8) ◽  
pp. 4244-4252
Author(s):  
H J Son ◽  
K Shahan ◽  
M Rodriguez ◽  
E Derman ◽  
F Costantini
Keyword(s):  
Transcription Initiation ◽  
Fusion Gene ◽  
Submaxillary Gland ◽  
Regulatory Elements ◽  
Specific Pattern ◽  
Regulatory Sequence ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
Dna Fragment

The MUP1.5b gene was previously found to be expressed specifically in the submaxillary gland and at high levels when introduced into mice as a transgene including 4.7 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. To localize regulatory elements responsible for this tissue-specific pattern of expression, we tested the expression of three additional MUP1.5b transgenes including various amounts of 5'-flanking DNA. These experiments indicated that sequences between -1.85 and -3.46 kb from the transcription initiation site were required for high-level expression in the submaxillary gland. The presence of regulatory elements in this region was also suggested by the detection of a DNase I-hypersensitive site, seen only in submaxillary gland nuclei, at position -2.5 kb upstream from the MUP1.5a gene, a member of the same MUP gene subfamily and virtually identical to the MUP1.5b gene. Further evidence for enhancer activity was provided by the ability of the 1.6-kb DNA fragment including sequences between -1.85 and -3.46 kb to stimulate the expression of an otherwise inactive MUP1.5b-chloramphenicol acetyltransferase fusion gene specifically in the submaxillary gland. The nucleotide sequence of this 1.6-kb DNA fragment was found to be identical for the MUP1.5a and MUP1.5b genes. Together, these results provide the first localization of a cis-acting regulatory sequence involved in the differential tissue-specific expression of the MUP gene family.

scholarly journals A gene-editing/complementation strategy for tissue-specific lignin reduction while preserving biomass yield

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hasi Yu ◽  
Chang Liu ◽  
Richard A. Dixon
Keyword(s):  
Gene Editing ◽  
Biomass Yield ◽  
Lignin Content ◽  
Open Reading Frame ◽  
General Strategy ◽  
Loss Of Function ◽  
Tissue Specific ◽  
Knock Out ◽  
Reading Frame ◽  
Endogenous Gene

Abstract Background Lignification of secondary cell walls is a major factor conferring recalcitrance of lignocellulosic biomass to deconstruction for fuels and chemicals. Genetic modification can reduce lignin content and enhance saccharification efficiency, but usually at the cost of moderate-to-severe growth penalties. We have developed a method, using a single DNA construct that uses CRISPR–Cas9 gene editing to knock-out expression of an endogenous gene of lignin monomer biosynthesis while at the same time expressing a modified version of the gene’s open reading frame that escapes cutting by the Cas9 system and complements the introduced mutation in a tissue-specific manner. Results Expressing the complementing open reading frame in vessels allows for the regeneration of Arabidopsis plants with reduced lignin, wild-type biomass yield, and up to fourfold enhancement of cell wall sugar yield per plant. The above phenotypes are seen in both homozygous and bi-allelic heterozygous T1 lines, and are stable over at least four generations. Conclusions The method provides a rapid approach for generating reduced lignin trees or crops with one single transformation event, and, paired with a range of tissue-specific promoters, provides a general strategy for optimizing loss-of-function traits that are associated with growth penalties. This method should be applicable to any plant species in which transformation and gene editing are feasible and validated vessel-specific promoters are available.

scholarly journals Molecular genetics of the Caenorhabditis elegans heterochronic gene lin-14.

Genetics ◽  
1989 ◽  
Vol 121 (3) ◽  
pp. 501-516 ◽  
Author(s):  
G Ruvkun ◽  
V Ambros ◽  
A Coulson ◽  
R Waterston ◽  
J Sulston ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
General Strategy ◽  
Cis Acting ◽  
Temporal Regulation ◽  
C Elegans ◽  
Dna Alterations ◽  
Heterochronic Gene ◽  
Systematic Identification

Abstract We describe a general strategy for the genetic mapping in parallel of multiple restriction fragment length polymorphism (RFLP) loci. This approach allows the systematic identification for cloning of physical genetic loci within about 100 kb of any gene in Caenorhabditis elegans. We have used this strategy of parallel RFLP mapping to clone the heterochronic gene lin-14, which controls the timing and sequence of many C. elegans postembryonic developmental events. We found that of about 400 polymorphic loci in the C. elegans genome associated with the Tc1 family of repetitive elements, six are within 0.3 map unit of lin-14. The three closest lin-14-linked Tc1-containing restriction fragments were cloned and used to identify by hybridization an 830-kb region of contiguous cloned DNA fragments assembled from cosmid and yeast artificial chromosome libraries. A lin-14 intragenic recombinant that separated a previously cryptic lin-14 semidominant mutation from a cis-acting lin-14 suppressor mutation was used to map the location of the lin-14 gene to a 25-kb region of this 830-kb contig. DNA probes from this region detected lin-14 allele-specific DNA alterations and a lin-14 mRNA. Two lin-14 semi-dominant alleles, which cause temporally inappropriate lin-14 gene activity and lead to the reiterated expression of specific early developmental events, were shown to delete sequences from the lin-14 gene and mRNA. These deletions may define cis-acting sequences responsible for the temporal regulation of lin-14.

scholarly journals All of the human β‐type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice

2009 ◽  
Vol 23 (12) ◽  
pp. 4335-4343 ◽  
Author(s):  
Eiichi Okamura ◽  
Hitomi Matsuzaki ◽  
Andrew D. Campbell ◽  
James Douglas Engel ◽  
Akiyoshi Fukamizu ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Enhancer Activity

Rescue of the lethal scl−/− phenotype by the human SCL locus

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3931-3938 ◽  
Author(s):  
Angus M. Sinclair ◽  
Anthony J. Bench ◽  
Adrian J. C. Bloor ◽  
Juan Li ◽  
Berthold Göttgens ◽  
...  
Keyword(s):  
Yeast Artificial Chromosome ◽  
Fetal Liver ◽  
Critical Role ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
Yolk Sac ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Helix Loop Helix

The stem cell leukemia (SCL) gene encodes a basic helix-loop-helix transcription factor with a critical role in the development of both blood and endothelium. Loss-of-function studies have shown that SCL is essential for the formation of hematopoietic stem cells, for subsequent erythroid development and for yolk sac angiogenesis. SCL exhibits a highly conserved pattern of expression from mammals to teleost fish. Several murine SCLenhancers have been identified, each of which directs reporter gene expression in vivo to a subdomain of the normal SCL expression pattern. However, regulatory elements necessary for SCL expression in erythroid cells remain to be identified and the size of the chromosomal domain needed to support appropriate SCL transcription is unknown. Here we demonstrate that a 130-kilobase (kb) yeast artificial chromosome (YAC) containing the human SCL locus completely rescued the embryonic lethal phenotype ofscl−/− mice. Rescued YAC+scl−/− mice were born in appropriate Mendelian ratios, were healthy and fertile, and exhibited no detectable abnormality of yolk sac, fetal liver, or adult hematopoiesis. The human SCL protein can therefore substitute for its murine homologue. In addition, our results demonstrate that the human SCL YAC contains the chromosomal domain necessary to direct expression to the erythroid lineage and to all other tissues in which SCL performs a nonredundant essential function.

scholarly journals Localization of Distant Urogenital System-, Central Nervous System-, and Endocardium-Specific Transcriptional Regulatory Elements in the GATA-3 Locus

1999 ◽  
Vol 19 (2) ◽  
pp. 1558-1568 ◽  
Author(s):  
Ganesh Lakshmanan ◽  
Ken H. Lieuw ◽  
Kim-Chew Lim ◽  
Yi Gu ◽  
Frank Grosveld ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Yeast Artificial Chromosome ◽  
Transgenic Animals ◽  
Regulatory Elements ◽  
Urogenital System ◽  
Specific Expression ◽  
Tissue Specific ◽  
Transcriptional Regulatory Elements ◽  
Transcriptional Regulatory

ABSTRACT We found previously that neither a 6-kbp promoter fragment nor even a 120-kbp yeast artificial chromosome (YAC) containing the whole GATA-3 gene was sufficient to recapitulate its full transcription pattern during embryonic development in transgenic mice. In an attempt to further identify tissue-specific regulatory elements modulating the dynamic embryonic pattern of the GATA-3 gene, we have examined the expression of two much larger (540- and 625-kbp) GATA-3 YACs in transgenic animals. A lacZ reporter gene was first inserted into both large GATA-3 YACs. The transgenic YAC patterns were then compared to those of embryos bearing the identical lacZinsertion in the chromosomal GATA-3 locus (creating GATA-3/lacZ “knock-ins”). We found that most of the YAC expression sites and tissues are directly reflective of the endogenous pattern, and detailed examination of the integrated YAC transgenes allowed the general localization of a number of very distant transcriptional regulatory elements (putative central nervous system-, endocardium-, and urogenital system-specific enhancers). Remarkably, even the 625-kbp GATA-3 YAC, containing approximately 450 kbp and 150 kbp of 5′ and 3′ flanking sequences, respectively, does not contain the full transcriptional regulatory potential of the endogenous locus and is clearly missing regulatory elements that confer tissue-specific expression to GATA-3 in a subset of neural crest-derived cell lineages.

scholarly journals Impact of broad regulatory regions on Gdf5 expression and function in knee development and susceptibility to osteoarthritis

2018 ◽  
Vol 77 (3) ◽  
pp. 450-450 ◽  
Author(s):  
Steven K Pregizer ◽  
Ata M Kiapour ◽  
Mariel Young ◽  
Hao Chen ◽  
Michael Schoor ◽  
...  
Keyword(s):  
Artificial Chromosome ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Loss Of Function ◽  
Regulatory Regions ◽  
Control Knee ◽  
And Function ◽  
Inactivating Mutation ◽  
Femoral Condyles

ObjectivesGiven the role of growth and differentiation factor 5 (GDF5) in knee development and osteoarthritis risk, we sought to characterise knee defects resulting from Gdf5 loss of function and how its regulatory regions control knee formation and morphology.MethodsThe brachypodism (bp) mouse line, which harbours an inactivating mutation in Gdf5, was used to survey how Gdf5 loss of function impacts knee morphology, while two transgenic Gdf5 reporter bacterial artificial chromosome mouse lines were used to assess the spatiotemporal activity and function of Gdf5 regulatory sequences in the context of clinically relevant knee anatomical features.ResultsKnees from homozygous bp mice (bp/bp) exhibit underdeveloped femoral condyles and tibial plateaus, no cruciate ligaments, and poorly developed menisci. Secondary ossification is also delayed in the distal femur and proximal tibia. bp/bp mice have significantly narrower femoral condyles, femoral notches and tibial plateaus, and curvier medial femoral condyles, shallower trochlea, steeper lateral tibial slopes and smaller tibial spines. Regulatory sequences upstream from Gdf5 were weakly active in the prenatal knee, while downstream regulatory sequences were active throughout life. Importantly, downstream but not upstream Gdf5 regulatory sequences fully restored all the key morphological features disrupted in the bp/bp mice.ConclusionsKnee morphology is profoundly affected by Gdf5 absence, and downstream regulatory sequences mediate its effects by controlling Gdf5 expression in knee tissues. This downstream region contains numerous enhancers harbouring human variants that span the osteoarthritis association interval. We posit that subtle alterations to morphology driven by changes in downstream regulatory sequence underlie this locus’ role in osteoarthritis risk.

scholarly journals Structure of the Chromosome VII Centromere Region in Neurospora crassa: Degenerate Transposons and Simple Repeats

1998 ◽  
Vol 18 (9) ◽  
pp. 5465-5477 ◽  
Author(s):  
Edward B. Cambareri ◽  
Rafael Aisner ◽  
John Carbon
Keyword(s):  
Neurospora Crassa ◽  
Yeast Artificial Chromosome ◽  
Fragment Length Polymorphism ◽  
Artificial Chromosome ◽  
Polymorphism Analysis ◽  
Dna Repeats ◽  
Centromere Region ◽  
Simple Repeats ◽  
Restriction Fragment Length ◽  
Repeat Induced Point Mutation

ABSTRACT DNA from the centromere region of linkage group (LG) VII ofNeurospora crassa was cloned previously from a yeast artificial chromosome library and was found to be atypical ofNeurospora DNA in both composition (AT rich) and complexity (repetitive). We have determined the DNA sequence of a small portion (∼16.1 kb) of this region and have identified a cluster of three new retrotransposon-like elements as well as degenerate fragments from the 3′ end of Tad, a previously identified LINE-like retrotransposon. This region contains a novel full-length but nonmobilecopia-like element, designated Tcen, that is only associated with centromere regions. Adjacent DNA contains portions of a gypsy-like element designated Tgl1. A third new element, Tgl2, shows similarity to theTy3 transposon of Saccharomyces cerevisiae. All three of these elements appear to be degenerate, containing predominantly transition mutations suggestive of the repeat-induced point mutation (RIP) process. Three new simple DNA repeats have also been identified in the LG VII centromere region. While Tcenelements map exclusively to centromere regions by restriction fragment length polymorphism analysis, the defective Tad elements appear to occur most frequently within centromeres but are also found at other loci including telomeres. The characteristics and arrangement of these elements are similar to those seen in theDrosophila centromere, but the relative abundance of each class of repeats, as well as the sequence degeneracy of the transposon-like elements, is unique to Neurospora. These results suggest that the Neurospora centromere is heterochromatic and regional in character, more similar to centromeres of Drosophila than to those of most single-cell yeasts.

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