Kb Of Upstream Sequence Research Articles

The Promoter and Multiple Enhancers of the pou4f3 Gene Regulate Expression in Inner Ear Hair Cells.

Few enhancers that target gene expression to inner ear hair cells (HCs) have been identified. Using transgenic analysis of enhanced green fluorescent protein (eGFP) reporter constructs and bioinformatics, we evaluated the control of pou4f3 gene expression, since it is expressed only in HCs within the inner ear and continues to be expressed throughout life. An 8.5-kb genomic DNA fragment 5' to the start codon, containing three regions of high cross-species homology, drove expression in all embryonic and neonatal HCs, and adult vestibular and inner HCs, but not adult outer HCs. Transgenes with 0.4, 0.8, 2.5, or 6.5kb of 5' DNA did not produce HC expression. However, addition of the region from 6.5 to 7.2kb produced expression in vestibular HCs and neonatal basal turn outer HCs, which also implicated the region from 7.2 to 8.5kb in inner and apical outer HC expression. Deletion of the region from 0.4 to 5.5kb 5' from the 8.5-kb construct did not affect HC expression, further indicating lack of HC regulatory elements. When the region from 1 to 0.4kb was replaced with the minimal promoter of the Ela1 gene, HC expression was maintained but at a drastically reduced level. Bioinformatics identified regions of highly conserved sequence outside of the 8.5kb, which contained POU4F3-, GFI1-, and LHX3-binding sites. These regions may be involved in maintaining POU4F3 expression in adult outer HCs. Our results identify separate enhancers at various locations that direct expression to different HC types at different ages and determine that 0.4kb of upstream sequence determines expression level. These data will assist in the identification of mutations in noncoding, regulatory regions of this deafness gene.

A 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long-term expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

Helper virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100bp fragments that each support long-term expression, and are contained within an ~320bp fragment located ~3kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100bp fragments contained 44 or 23bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42bp sequence, centered on the 23bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16bp maximum essential sequence. This 16bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.

Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11

The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5' proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3' hepatic control region, derived from a region approximately 18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3' splice acceptor sites causing deletion of cloned 5' untranslated mRNA sequences and, in some cases, deletion of the 5' end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3' splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1-exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression.

Long-term Physiologically Regulated Expression of the Low-density Lipoprotein Receptor In Vivo Using Genomic DNA Mini-gene Constructs

Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH.

An Iron Enhancer Element in the FTN-1 Gene Directs Iron-dependent Expression in Caenorhabditis elegans Intestine

Ferritin is a ubiquitous protein that sequesters iron and protects cells from iron toxicity. Caenorhabditis elegans express two ferritins, FTN-1 and FTN-2, which are transcriptionally regulated by iron. To identify the cis-acting sequences and proteins required for iron-dependent regulation of ftn-1 and ftn-2 expression, we generated transcriptional GFP reporters corresponding to 5 '-upstream sequences of the ftn-1 and ftn-2 genes. We identified a conserved 63-bp sequence, the iron-dependent element (IDE), that is required for iron-dependent regulation of a ftn-1 GFP reporter in intestine. The IDE contains two GATA-binding motifs and three octameric direct repeats. Site-directed mutagenesis of the GATA sequences, singly or in combination, reduces ftn-1 GFP reporter expression in the intestine. In vitro DNA mobility shift assays show that the intestine-specific GATA protein ELT-2 binds to both GATA sequences. Inhibition of ELT-2 function by RNA interference blocks ftn-1 GFP reporter expression in vivo. Insertion of the IDE into the promoter region of a heterologous reporter activates iron-dependent transcription in intestine. These data demonstrate that the activation of ftn-1 and ftn-2 transcription by iron requires ELT-2 and that the IDE functions as an iron-dependent enhancer in intestine.

Molecular Actions of 1,25-Dihydroxyvitamin D3 on Genes Involved in Calcium Homeostasis

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] functions in vertebrate organisms as a primary regulator of calcium and phosphorus homeostasis, an activity that is achieved through direct actions on gene expression in intestine, kidney, and bone. Recent studies have identified novel genes such as TRPV5, TRPV6, and RANKL whose products are integral to the maintenance of extracellular calcium. The objective of this progress report/review is to describe our recent results that identify the mechanisms of 1,25(OH)(2)D(3) action on the expression of TRPV6 and RANKL. A series of molecular, cellular, and in vivo studies have been conducted to define the molecular mechanisms that control the expression of TRPV6 and RANKL. Cell culture-based assays, chromatin immunoprecipitation (ChIP) and ChIP-DNA microarray (ChIP-chip) methods, and a series of molecular techniques were used to identify and characterize upstream regions of mouse and human TRPV6 and RANKL genes. We discovered that these genes were regulated by at least five separate enhancer regions. In the TRPV6 gene, these enhancers were all located within 5 kb of the transcriptional start site (TSS), and each contained one or more vitamin D regulatory elements (VDREs). In the RANKL gene, these regulatory regions span over 80 kb of upstream sequence, the most distal 76 kb from the TSS. This regulatory region is central to the regulation of RANKL expression in vitro and in vivo. Our studies identified key regulatory regions within the TRPV6 and RANKL genes that are essential for their individual expression in the intestine and bone, respectively.

Imprinting and gene silencing in mice and Drosophila.

H19 and Igf2 are located within a large imprinting domain that confers monoallelic silencing of parental alleles. The silent paternal allele of H19 is hypermethylated and relatively resistant to nucleases. Using a 130 kb yeast artificial chromosome clone, appropriate imprinting of both H19 and Igf2 was observed at single insert loci in transgenic mice. Imprinting was also observed for H19-lacZ transgenes containing 4 kb of upstream sequence, but only at multicopy loci. The H19 RNA is therefore not essential for imprinting. When the H19-lacZ transgene was introduced into Drosophila, a 1.2 kb region was identified within the 4 kb upstream flank that functioned as a bi-directional silencer. This cis element is located within a region that is apparently necessary for imprinting in mice. These studies suggest an evolutionarily conserved mechanism for gene silencing in Drosophila and imprinting in mice. We propose a new model for imprinting of H19 and Igf2 in mice in which silencing of H19 is the default state, and activation of the maternal allele requires a specific activator element.

A Transgenic Mouse Model To Study JAK2-V617F In Vivo .

It is generally assumed that the JAK2-V617F mutation is the cause of disease in a large proportion of patients with myeloproliferative disorders. However, recent evidence suggests that additional genetic alterations might be collaborating with JAK2-V617F. Most importantly, screenings of hematopoietic cells from JAK2-V617F positive patients using quantitative PCR-based methods have revealed a strong variability in the fraction of cells that carry the mutation and in some patients a striking discrepancy with the extent of clonal hematopoiesis. On the other hand, retroviral overexpression of JAK2-V617F in mouse bone marrow transplantation models causes phenotypes that resemble human polycythemia vera, the MPD entity in which the incidence of JAK2-V617F is highest. In the mouse transplantation models described to date, retroviral vectors were used that bear the risk of insertional mutagenesis and in most cases lead to a strong overexpression of JAK2-V617F. These limitations might hamper an accurate reproduction of the human disease. To express the mutant JAK2 at physiological levels in a mouse model, we generated JAK2-V617F transgenic mice. The transgene was constructed based on a human BAC containing JAK2 exons 1–12 and approximately 100 kb of upstream sequence. A cDNA fragment covering exons 13–25 of JAK2, which includes the V617F mutation, was appended by homologous recombination in bacteria. We also generated an identical construct with the JAK2 wild type sequence as a control. Pronuclear microinjection of the control construct yielded 8 viable and fertile founders with transgene copy numbers ranging from 1 to 18. We also obtained 3 mice that were positive for the JAK2-V617F transgene. One of these mice died 2 days after birth, a second mouse died at the age of 5 weeks. This mouse had 3 integrated copies of the transgene and interestingly an approximately 10-fold enlarged spleen. Unfortunately, we were unable to obtain additional data from this mouse. The third mouse with one integrated copy of the transgene was viable and gave rise to a transgenic line (VF1). The VF1 line expresses the transgene at approximately 25% of the endogenous wild-type Jak2 level. We analyzed blood counts at the age of 5–6 weeks and observed a small but significant elevation of platelet numbers (approximately 1.2-fold), which was reproduced when transplanting JAK2-VF1 bone marrow into wild type recipients. No changes in hematocrit or white blood cell count were detected. The low yield of JAK2-V617F transgenic mice together with the early death of two of the founders suggested that the transgene could be lethal when expression exceeds a certain level. To overcome this potential problem and to allow for inducible expression of the JAK2-V617F transgene, we generated a second construct in which exons 13–25 of the original transgene were inverted to abolish expression of catalytically active Jak2 in this configuration. Antiparallel loxP sites were placed at the borders of this inverted fragment to allow Cre-mediated repair of the transgene. These loxP sites contain previously characterized mutations to prevent continuous recombination. We have obtained 4 founders of this Cre-activated JAK2-V617F transgene with copy numbers between 1 and 9. After crossing to Mx-Cre mice and inducing Cre we observed repair of the transgene at the DNA level. Furthermore, mRNA of the recombined JAK2-V617F allele was detectable in peripheral blood. The effects of the JAK2-V617F transgene induction on the hematopoietic system are currently being studied in detail and will be presented.

Comparative Genomic Analysis of the 5′-Upstream Sequences of von Willebrand Factor and 27 Other Endothelial-Specific Genes Identifies Conserved Noncoding Sequences That Are DNase Hypersensitive.

Von Willebrand factor (VWF), a key blood clotting protein, is a standard histologic marker for endothelial origin. Previously published studies characterizing tissue-specific regulatory elements have concentrated on the proximal promoter region (between −2,645 and the end of murine Vwf intron 1). Transgenic mouse models using these sequences have found elements needed for VWF expression in some, but not all, populations of endothelial cells. To identify potential novel VWF distal regulatory elements, as well as sequences contributing to the more general endothelial-specific expression program, we aligned human and mouse orthologous genomic sequences from VWF and 27 other endothelial-specific genes, extending from the start of transcription to the neighboring 5′-upstream gene (up to 100 kb of sequence), excluding the core promoter, and including intron 1. We analyzed the resulting 262 endothelial cell-specific gene conserved noncoding sequences (EC-CNSs) using a specific quantitative PCR assay for DNase I hypersensitive sites, an indicator of an open chromatin state which suggests the presence of a transcription regulatory complex. 33% of the EC-CNSs (including 6 of 9 upstream of VWF) were hypersensitive (HS) in human umbilical vein endothelial cells (HUVECs), while only 16% were HS in control human fibroblast cells. 20% of the EC-CNSs were HS in HUVECs only, suggesting that they may identify novel endothelial-specific transcriptional elements. When the 262 EC-CNSs were compared to CNSs from non-endothelial genes, a single motif, AGGAA(A/G), was identified as overrepresented in EC-CNSs. This association was replicated in an independent set of 28 additional genes with high HUVEC expression. The motif was also overrepresented in the subset of HS EC-CNSs. The AGGAA(A/G) motif includes the core binding sequence [GGA(A/T)] of the Ets family of transcription factors, which contains several members implicated in tissue-specific gene expression. These results suggest that the AGGAA(A/G) motif may represent the target sequence for a specific Ets family member critical for determining the endothelial-specific gene expression program. We are currently testing this hypothesis by analyzing this motif in Vwf BAC transgenic mice containing 177 kb of upstream sequence, including all ten identified EC-CNSs. Additionally, we have expanded our analysis of the VWF genomic region to include over 700 kb of sequence surrounding and including the gene, searching for additional EC-CNSs conserved across eleven mammalian species. This comparative genomic approach, coupled with functional analysis of putative regulatory elements, should provide new insight into the endothelial cell gene expression program and the more general problem of tissue-specific gene regulation.

Upstream and coding region CYP2C9 polymorphisms

To assess whether CYP2C9 alleles other than CYP2C9*2 and *3 are associated with a low-warfarin dose requirement and the relevance of upstream CYP2C9 polymorphisms to dose requirement and metabolism. CYP2C9 exons, intron-exon boundaries and 3 kb of upstream sequence in 20 patients requiring <or= 1.5 mg warfarin per day and with apparently homozygous wild-type or heterozygous CYP2C9*2 genotypes were screened for novel polymorphisms by single-strand conformational polymorphism analysis. PCR-based genotyping assays for novel upstream and other known polymorphisms were used to screen a larger patient population of known CYP2C9*2 and *3 genotype requiring a range of warfarin doses. Polymorphisms at eight different upstream sites were found, five of which were already described. We found that the majority of the upstream polymorphisms were in complete linkage disequilibrium with previously described coding region polymorphisms. However, two polymorphisms, T-1188C and the novel DeltaG-2664DeltaT-2665, occurred both in individuals who were otherwise wild-type and in individuals positive for coding region polymorphisms. Evidence for 11 haplotypes, including 8 with frequencies >or= 0.01, was obtained. In individuals negative for coding region polymorphisms, neither individual genotypes for T-1188C or DeltaG-2664DeltaT-2665 or particular combinations of haplotype pairs were predictive of dose requirement or S-warfarin total clearance, suggesting neither upstream polymorphism was functionally significant. Dose requirements in CYP2C9*11 heterozygotes were not statistically significantly different from homozygous wild-type individuals. The coding region non-synonymous polymorphisms associated with the CYP2C9*2 and CYP2C9*3 alleles are the major CYP2C9-related factor affecting warfarin dose in UK Caucasians. Upstream CYP2C9 polymorphisms do not appear to be important independent determinants of dose requirement.

Cloning and initial characterization of the human cardiac sodium channel (SCN5A) promoter.

Despite the primacy of the sodium current in cardiac electrophysiology and evidence that decreased sodium current is arrhythmogenic in humans, little is known about transcriptional regulation of the underlying gene, SCN5A. We have cloned a 2.7 kb segment of 5'-flanking region of SCN5A and identified multiple transcription initiation sites by primer extension and RNase protection. Transient transfection assays in neonatal mouse myocytes and in Chinese Hamster Ovary (CHO) cells were employed to identify promoter activities. PCR-single stranded conformational polymorphism (SSCP) analysis was used to screen DNA variants in the promoter region. The fragment includes >2 kb of upstream sequence, the 173-bp non-coding exon 1, and a portion of the 16-kb intron 1; the region is highly GC-rich and TATA-less. Transient transfection assays in neonatal mouse myocytes and in CHO cells identified (1) a core promoter in the -261/+140 segment, (2) regions conferring approximately 3-fold decreases from core promoter activity in the 5' upstream region (-261/-454 and -1020/-2109), and approximately 3-fold increases in intron 1 (+255/+410 and+539/+613), and (3) a very strong negative regulatory region between +613 and +754 in intron 1. A core promoter polymorphism, present in 6/142 (4%) of normal alleles screened, increased reporter activity approximately 50% in myocytes but not in CHO cells. The SCN5A promoter includes multiple positive and negative cis-acting elements extending into intron 1. A common polymorphism in this region modulates channel expression in vitro.

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation.

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged approximately 35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

Transgenic Mice Demonstrate Novel Promoter Regions for Tissue-Specific Expression of the Urokinase Receptor Gene

The urokinase-type plasminogen activator receptor (u-PAR) contributes to cell migration and proteolysis in normal and cancerous tissues. Currently, there are no reports on the regulatory regions directing tissue-specific expression. Consequently, we undertook a study to identify novel promoter regions required for expression of this gene in transgenic mice bearing a LacZ reporter regulated by varying amounts (0.4, 1.5, and 8.5 kb) of upstream sequence. The 0.4-kb u-PAR upstream sequence directed weak and strong LacZ expression in the placenta and epididymis, respectively, both of which are tissues that express endogenous u-PAR. Conversely, transgene expression in the apical cells of the colon positive for endogenous u-PAR protein required 1.5 kb of upstream sequence for optimal expression. Furthermore, chromatin accessibility assays coupled with real-time polymerase chain reaction suggested a putative regulatory region spanning -1295/-1192 driving u-PAR expression in colonic cells. Interestingly, placental transgene expression was augmented with the 8.5-kb upstream fragment compared with the shorter 1.5-kb fragment indicating contributing element(s) between -1.5 and -8.5 kb. Thus, while 0.4 kb of upstream sequence directs u-PAR expression in the epididymis, sequences located between -0.4 and -1.5 kb and between -1.5 and -8.5 kb are required for optimal tissue-specific expression in the colon and the placenta, respectively.

3P-0749 Impact of T-1131C polymorphism within A5 gene on the small, dense LDL concentration in healthy Korean men and women

The cytochrome P450 enzyme, cholesterol 7a-hydroxylase (CYP7A), catalyses the first and rate-limiting step in the conversion of cholesterol to bile acids. Expression of the CYP7A gene is under complex physiological control, encompassing amongst others a feedback down-regulation by bile acids. Using the CYP7A cDNA of the rat as a probe, we isolated a rat genomic clone containing the 5' part of the gene, including approximately 3.6 kb of upstream sequences. Sequence analysis revealed the presence of several putative regulatory elements. Transient expression analyses of transfected primary hepatocytes demonstrated that the major transcription-activating region is located in the proximal 145 nucleotide (nt). Upon addition of taurocholate to the culture, a significant reduction of the transcriptional activity was observed, suggesting the presence of a bile acid-responsive element in the proximal region of the CYP7A promoter. In addition, evidence was obtained for the presence of a thyroxine-responsive site further upstream. After addition of taurocholate, steady-state CYP7A mRNA levels, as judged by Northern analysis of hepatocyte RNA, are eightfold reduced. On the other hand, the transcriptional activity of CYP7A, as shown both in CAT assays and run-on experiments, revealed only a threefold decrease. These experiments suggest that both transcriptional control and regulation of CYP7A mRNA stability play an important part in the feedback regulation of CYP7A activity in the rat.

Analysis of Transcriptional Regulatory Sequences of the N-Methyl-d-aspartate Receptor 2A Subunit Gene in Cultured Cortical Neurons and Transgenic Mice

The postnatal appearance and up-regulation of the NR2A subunit of the N-methyl-d-aspartate receptor contributes to the functional heterogeneity of the receptor during development. To elucidate the molecular mechanisms that regulate the neural and developmental specific expression of NR2A, an upstream approximately 9-kb region of the gene harboring the promoter was isolated and characterized in transgenic mice and transfected cortical neurons. Transgenic mouse lines generated with luciferase reporter constructs driven by either 9 or 1 kb of upstream sequence selectively transcribe the transgene in brain, as compared with other non-neural tissues. Reporter luciferase levels in dissociated cultures made from these mice are over 100-fold greater in neuronal/glial co-cultures than in pure glial cultures. Analysis of NR2A 5'-nested deletions in transfected cultures of cortical neurons and glia indicate that while sequences residing upstream of -1079 bp augment NR2A neuronal expression, sequences between -486 and -447 bp are sufficient to maintain neuronal preference. An RE1/NRSE element is not necessary for NR2A neuron specificity. Furthermore, comparison of the 5'-deletion constructs in cortical neurons grown for 5, 8, 11, or 14 days in vitro indicate that sequences between -1253 and -1180 bp are necessary for maturational up-regulation of NR2A. Thus, different cis-acting sequences control the regional and temporal expression of NR2A, implicating distinct regulatory pathways.

Biochemical and molecular characterization of two cytidine deaminases in the nematode Caenorhabditis elegans.

Two cytidine deaminases (CDDs) from the free-living nematode Caenorhabditis elegans have been cloned and characterized. Both Ce-CDD-1 and Ce-CDD-2 are authentic deaminases and both exhibit RNA-binding activity towards AU-rich templates. In order to study their temporal and spatial expression patterns in the worm, reporter gene constructs were made using approx. 2 kb of upstream sequence. Transfection of C. elegans revealed that both genes localized to the cells of the intestine, although their temporal expression patterns were different. Expression of Ce-cdd-1 peaked in the early larval stages, whereas Ce-cdd-2 was expressed in all life cycle stages examined. RNA-interference (RNAi) assays were performed for both genes, either alone or in combination, but only cdd-2 RNAi produced a consistent visible phenotype. A proportion of eggs laid from these worms were swollen and distorted in shape.

Characterization of genomic DNA encoding cecropins from an Aedes albopictus mosquito cell line

We used cDNA probes from Aedes albopictus mosquito cecropins AalCecA, B, and C to obtain genomic DNA copies and flanking DNA. Two gene copies (AalCecA1 and A2, AalCecB1 and B2, AalCecC1 and C2) encoding each of the three mature cecropin peptides were recovered. All these genes had a similar organization, into two exons interrupted by a single short intron. AalCecA1 and AalCecA2 encode mature protein products that differ by one amino acid residue, while AalCecB1 and AalCecB2, AalCecC1 and AalCecC2 encode identical mature cecropin peptides, respectively. The AalCecB and C gene pairs each share a common intergenic region of approximately 1 kb, with the two coding regions transcribed in opposite directions. With the exception of small insertions/deletions, the intergenic spacer region was highly conserved between the B1/C1 and B2/C2 clones. In transfected cells, 0.8 kb of upstream sequence was sufficient for inducible expression of AalCecA1. Within this region, a 28 bp sequence at positions -192 to -165 upstream of the transcription initiation site was found to contain a potential regulatory element. In electrophoretic mobility shift assays, synthetic double-stranded DNA containing this 28 bp sequence retarded protein in cytoplasmic and nuclear extracts from C7-10 cells.

A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development.

The muscle-specific transcription factors Myf5 and Mrf4 are two of the four myogenic regulatory factors involved in the transcriptional cascade responsible for skeletal myogenesis in the vertebrate embryo. Myf5 is the first of these four genes to be expressed in the mouse. We have previously described discrete enhancers that drive Myf5 expression in epaxial and hypaxial somites, branchial arches and central nervous system, and argued that additional elements are required for proper expression (Summerbell, D., Ashby, P. R., Coutelle, O., Cox, D., Yee, S. P. and Rigby, P. W. J. (2000) Development 127, 3745-3757). We have now investigated the transcriptional regulation of both Myf5 and Mrf4 using bacterial artificial chromosome transgenesis. We show that a clone containing Myf5 and 140 kb of upstream sequences is sufficient to recapitulate the known expression patterns of both genes. Our results confirm and reinforce the conclusion of our earlier studies, that Myf5 expression is regulated differently in each of a considerable number of populations of muscle progenitors, and they begin to illuminate the evolutionary origins of this complex regulation. We further show that separate elements are involved in the activation and maintenance of expression in the various precursor populations, reflecting the diversity of the signals that control myogenesis. Mrf4 expression requires at least four elements, one of which may be shared with Myf5, providing a possible explanation for the linkage of these genes throughout vertebrate phylogeny. Further complexity is revealed by the demonstration that elements which control Mrf4 and Myf5 are embedded in an unrelated neighbouring gene.

A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development.

The muscle-specific transcription factors Myf5 and Mrf4 are two of the four myogenic regulatory factors involved in the transcriptional cascade responsible for skeletal myogenesis in the vertebrate embryo. Myf5 is the first of these four genes to be expressed in the mouse. We have previously described discrete enhancers that drive Myf5 expression in epaxial and hypaxial somites, branchial arches and central nervous system, and argued that additional elements are required for proper expression (Summerbell, D., Ashby, P. R., Coutelle, O., Cox, D., Yee, S. P. and Rigby, P. W. J. (2000) Development 127, 3745-3757). We have now investigated the transcriptional regulation of both Myf5 and Mrf4 using bacterial artificial chromosome transgenesis. We show that a clone containing Myf5 and 140 kb of upstream sequences is sufficient to recapitulate the known expression patterns of both genes. Our results confirm and reinforce the conclusion of our earlier studies, that Myf5 expression is regulated differently in each of a considerable number of populations of muscle progenitors, and they begin to illuminate the evolutionary origins of this complex regulation. We further show that separate elements are involved in the activation and maintenance of expression in the various precursor populations, reflecting the diversity of the signals that control myogenesis. Mrf4 expression requires at least four elements, one of which may be shared with Myf5, providing a possible explanation for the linkage of these genes throughout vertebrate phylogeny. Further complexity is revealed by the demonstration that elements which control Mrf4 and Myf5 are embedded in an unrelated neighbouring gene.

Cloning and sequence of the gene encoding the muscle fatty acid binding protein from the desert locust, Schistocerca gregaria

Muscle fatty acid binding protein (FABP) is a major cytosolic protein in flight muscle of the desert locust, Schistocerca gregaria. FABP expression varies greatly during development and periods of increased fatty acid utilization, but the molecular mechanisms that regulate its expression are not known. In this study, the gene coding for locust muscle FABP was amplified by PCR and cloned, together with 1.2 kb of upstream sequence. The sequence coding for the 607 bp cDNA is interrupted by two introns of 12.7 and 2.9 kb, inserted in analogous positions as the first and third intron of the mammalian homologues. Both introns contain repetitive sequences also found in other locust genes, and the second intron contains a GT-microsatellite. The promoter sequence includes a canonical TATA box 24 bp upstream of the transcription start site. The upstream sequence contains various potential myocyte enhancer sequences and a 160 bp segment that is repeated three times. In database searches in the genome database of Drosophila melanogaster, a gene with the same gene organization and promoter structure was identified, likely the dipteran homologue of muscle FABP. Upstream of both insect genes, a conserved 19 bp inverted repeat sequence was detected. A similar but reverse palindrome is also present upstream of all mammalian heart FABP genes, possibly representing a novel element involved in muscle FABP expression.