Role of the Agrobacterium tumefaciens VirD2 Protein in T-DNA Transfer and Integration

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called ω that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the ω region are not essential for nuclear uptake of T-DNA, and further suggested that the ω domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the ω domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of β-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid (X-gluc). However, using an ω-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 ω deletion/substitution. Taken together, these data indicate that the VirD2 ω domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the ω mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an ω mutation. In addition, a mutant VirD2 protein lacking the ω domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the ω and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the ω domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

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Natural Zeitgebers Under Temperate Conditions Cannot Compensate for the Loss of a Functional Circadian Clock in Timing of a Vital Behavior in Drosophila

Journal of Biological Rhythms ◽

10.1177/0748730421998112 ◽

2021 ◽

pp. 074873042199811

Author(s):

Franziska Ruf ◽

Oliver Mitesser ◽

Simon Tii Mungwa ◽

Melanie Horn ◽

Dirk Rieger ◽

...

Keyword(s):

Molecular Clock ◽

Time Window ◽

Fruit Fly ◽

Diel Activity ◽

Wild Type ◽

Statistical Measures ◽

Clock Mutant ◽

Full Complement ◽

The Right

The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period01 mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period01 mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling ( Pdf01 and han5304 PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling

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GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8565-8574.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8565-8574 ◽

Cited By ~ 20

Author(s):

Anthony J. Greenberg ◽

Paul Schedl

Keyword(s):

Fusion Protein ◽

Transcriptional Activation ◽

Functional Difference ◽

Wild Type ◽

Gaga Factor ◽

Phenotypic Analysis ◽

Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

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Agrobacterium-Mediated Transformation of Aspergillus awamori in the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

Journal of Bacteriology ◽

10.1128/jb.186.7.2038-2045.2004 ◽

2004 ◽

Vol 186 (7) ◽

pp. 2038-2045 ◽

Cited By ~ 21

Author(s):

Caroline B. Michielse ◽

Arthur F. J. Ram ◽

Paul J. J. Hooykaas ◽

Cees A. M. J. J. van den Hondel

Keyword(s):

Single Copy ◽

Full Length ◽

Aspergillus Awamori ◽

Wild Type ◽

Dna Structures ◽

Dna Integration ◽

Virulence Proteins ◽

Type Transformation ◽

The Right ◽

Transposon Insertions

ABSTRACT Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.

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Agrobacterium tumefaciens Transformation of the Radiation Hypersensitive Arabidopsis thaliana Mutants uvh1 and rad5

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.11.1136 ◽

1998 ◽

Vol 11 (11) ◽

pp. 1136-1141 ◽

Cited By ~ 19

Author(s):

Jaesung Nam ◽

Kirankumar S. Mysore ◽

Stanton B. Gelvin

Keyword(s):

Arabidopsis Thaliana ◽

Agrobacterium Tumefaciens ◽

Transformation Process ◽

Stable Transformation ◽

Wild Type ◽

Dna Integration ◽

Agrobacterium Tumefaciens Transformation ◽

Inoculation Assay

The Arabidopsis thaliana mutants uvh1 and rad5, originally identified as radiation hypersensitive, were reported to be deficient in T-DNA integration based on the relative efficiencies of stable transformation and T-DNA transfer. We reassessed these mutants for susceptibility to transformation by Agrobacterium tumefaciens. The mutant rad5 showed a significant reduction in the efficiency of transient as well as stable transformation, compared with its wild-type progenitor. These data indicate that rad5 is blocked at a step in the transformation process prior to T-DNA integration. We additionally found, using both an in vitro root inoculation and an in vivo flower bolt inoculation assay, that the mutant uvh1 is as susceptible to A. tumefaciens-mediated transformation as is its wild-type progenitor, C10.

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Corepression of the P1 Addiction Operon by Phd and Doc

Journal of Bacteriology ◽

10.1128/jb.180.23.6342-6351.1998 ◽

1998 ◽

Vol 180 (23) ◽

pp. 6342-6351 ◽

Cited By ~ 72

Author(s):

Roy Magnuson ◽

Michael B. Yarmolinsky

Keyword(s):

Fusion Protein ◽

Binding Sites ◽

Wild Type ◽

Dna Complexes ◽

Present Evidence ◽

Cooperative Interactions ◽

Regulatory Properties

ABSTRACT The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmid-free segregants, and Phd, an unstable antidote that neutralizes the toxin. Additionally, these products repress transcription of the operon. The antidote binds to two adjacent sites in the promoter. Here we present evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation,docH66Y. The DocH66Y protein retained the regulatory properties of the wild-type protein, but not its toxicity. In vivo, DocH66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Binding of toxin to antidote may be the physical basis for the neutralization of toxin. DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. Although DocH66Y enhanced the binding of Phd to two adjacent Phd-binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates cooperative interactions between adjacent Phd-binding sites. Several electrophoretically distinct protein-DNA complexes were observed with different amounts of DocH66Y relative to Phd. Maximal repression and specificity of DNA binding were observed with subsaturating amounts of DocH66Y relative to Phd. Analogous antidote-toxin pairs appear to have similar autoregulatory circuits. Autoregulation, by dampening fluctuations in the levels of toxin and antidote, may prevent the inappropriate activation of the toxin.

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Genome Wide Location Analysis Reveals Deregulated MicroRNA Genes In MLL Rearragned Leukemic Genome

Blood ◽

10.1182/blood.v116.21.2507.2507 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2507-2507

Author(s):

Shuangli Mi ◽

Fuhong He ◽

Jun Wu ◽

Jing Zhou ◽

George Wu ◽

...

Keyword(s):

Fusion Protein ◽

Mirna Expression ◽

Expression Profiling ◽

Fusion Proteins ◽

Target Genes ◽

Wild Type ◽

Protein Coding ◽

Protein Coding Genes ◽

Mrna Gene

Abstract Abstract 2507 The MLL (mixed-lineage leukemia) gene encodes a histone methyltransferase that is critical in maintaining gene expression during hematopoiesis. Chromosomal translocations disrupting MLL often leads to the creation of MLL fusion genes that act as potent drivers of acute leukemia. MLL fusion proteins are oncogenic transcription factors that activate the expression of downstream target genes. Expression profiling on patient primary samples and established mouse models has revealed hundreds of protein coding genes which are either up-regulated or down-regulated in MLL associated leukemias. Persistent coexpression of two of those genes, HoxA9 and Meis1, is essential for the initiation and maintenance of MLL leukemia. Our studies have also shown strong association of a microRNA (miRNA) expression signature with MLL- rearranged leukemia, and the expression of several miRNAs were under the control of MLL wild type and fusion proteins. Although profiling of miRNA expression has been reported, the mechanisms underlying deregulated miRNA expression in MLL associated leukemia are poorly understood. Given the role of miRNA as a global suppressor of mRNA gene expression, we hypothesized that the expression of miRNAs could be directly activated by MLL fusion and/or wild type proteins upon MLL gene rearrangement and subsequently down-regulate pertinent target mRNAs to contribute to leukemogenesis. To test our hypothesis in a systematic way, we examined an inducible MLL-ENL-ER transformed mouse cell line, which grow as myeloblastic cells in the presence of MLL-ENL, and differentiate into neutrophils upon inactivation of the fusion protein. Using chromatin immunoprecipitation assay followed by next generation sequencing (ChIP-Seq), we determined whole genome MLL binding pattern in this cellular model. Upon activation of MLL-ENL, 24 miRNAs showed a significant increase in the level of MLL binding (FDR<0.25), suggesting that those genes are directly bound by the MLL-ENL fusion protein. To explore the impact of MLL fusion protein on miRNA and mRNA gene regulation, we performed whole genome expression analysis using Affymetrix mouse microarray in the presence or absence of MLL-ENL. Upon induction of MLL-ENL, the expression levels of 38 miRNAs (out of 609 tiled on the array) were increased, and 57 of 7858 expressed protein-coding genes were down-regulated. An integrative analysis of MLL binding and mRNA/miRNA expression profiling data showed that transcription of three miRNAs were activated upon binding of MLL-ENL, and ten protein coding genes are potential targets of these miRNAs. We are currently exploring the role of these three miRNAs and their respective mRNA target genes in leukemogenesis using in vitro and in vivo models. Taken together, our data suggest that MLL fusion protein may play an important role in leukemogenesis by promoting miRNA transcription, which subsequently inhibit the expression of critical mRNA target genes. Our study provides a basis to further explore the regulatory network involving MLL fusion protein and its key miRNA target genes in the leukemic genome. Disclosures: No relevant conflicts of interest to declare.

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Association of A313g Glutathione S-Transferase P1 (GSTP1) Inborn Polymorphism with Susceptibility to De Novo MDS

Blood ◽

10.1182/blood.v120.21.1445.1445 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1445-1445

Author(s):

Sophia Zachaki ◽

Chryssa Stavropoulou ◽

Aggeliki Daraki ◽

Marina Kalomoiraki ◽

Panagoula Kollia ◽

...

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Type A ◽

Common Mechanism ◽

Genotype Distribution ◽

Distribution Analysis ◽

Wild Type ◽

Increased Risk ◽

Variant Genotype

Abstract Abstract 1445 Models for the pathogenesis of myelodysplastic syndromes (MDS) imply the role of individual genetic variations in genes involved in detoxification mechanisms. GSTP1 enzyme plays a key role in detoxification of a variety of electrophilic compounds, such as benzo [a]-pyrene and other polycyclic aromatic hydrocarbons (PAHs), chemotherapy drugs and products of oxidative stress. GSTP1 acts through a common mechanism of conjugating reactive oxygen species (ROS) with glutathione, enabling their detoxification and elimination and thus defending tissues against DNA damage. The corresponding gene is subject to a single-nucleotide polymorphism (A313G) leading to abolished enzyme activity. Thus, individuals homozygous for the variant G allele (G/G) have a lower conjugating activity than individuals homozygous for the wild type A allele (A/A), while heterozygotes (A/G) display intermediate activity. The aim of the present study was to evaluate whether the GSTP1 polymorphism influences susceptibility to MDS and/or promote specific chromosomal aberrations. We conducted a case-control study in 310 de novo MDS patients and 370 unrelated healthy controls using both a conventional PCR-RFLP assay and a novel Real-Time PCR genotyping method using hybridization probe technology. The GSTP1 gene status was also evaluated in relation to patients' characteristics and chromosomal abnormalities. Comparison of the genotype distribution between controls and MDS cases revealed a significantly higher frequency of the variant genotypes (heterozygotes A/A and homozygotes G/G) among MDS patients, as compared to controls (p<0.0001, χ2=31.167, df=2). The most marked statistical difference between MDS patients and controls was observed between the wild-type (A/A) and the homozygous variant genotype (G/G), since subjects carrying the G/G variant genotype showed a 4.1-fold increased risk of MDS prevalence than subjects carrying the wild-type A/A genotype (p=0.000, χ2=30.5, d.f.=1, OR=4.098, 95%CI=[2.433–6.897]). Allele frequencies distribution analysis between patients and controls, showed that MDS patients exhibited a 1.9-fold increased risk of carrying at least one variant G allele, as compared to the controls (p<0.0001, d.f.=1, OR =1.9, 95%CI=[1.48–2.34]). There was no association between the GSTP1 polymorphism and gender or any specific cytogenetic subgroup, while stratification of patients according to age showed a differential GSTP1 genotype distribution (p=0.007). Our results, derived from the larger series of primary MDS cases tested for the GSTP1 genetic background, reveal an increased incidence of the GSTP1 variant genotypes among MDS patients, providing evidence for a potential pathogenetic role of the GSTP1 polymorphism on de novo MDS risk. Disclosures: No relevant conflicts of interest to declare.

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Role of Trehalose Transport and Utilization in Sinorhizobium meliloti-Alfalfa Interactions

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0694 ◽

2005 ◽

Vol 18 (7) ◽

pp. 694-702 ◽

Cited By ~ 28

Author(s):

John Beck Jensen ◽

Osei Yaw Ampomah ◽

Richard Darrah ◽

N. Kent Peters ◽

T. V. Bhuvaneswari

Keyword(s):

Agrobacterium Tumefaciens ◽

Sinorhizobium Meliloti ◽

Brucella Melitensis ◽

Sole Source ◽

Root Surface ◽

Wild Type ◽

Major Pathway ◽

Mesorhizobium Loti ◽

Infection Threads

Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rm1021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.

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Activation tagging of the LEAFY PETIOLE gene affects leaf petiole development in Arabidopsis thaliana

Development ◽

10.1242/dev.127.22.4971 ◽

2000 ◽

Vol 127 (22) ◽

pp. 4971-4980 ◽

Cited By ~ 1

Author(s):

E. van der Graaff ◽

A.D. Dulk-Ras ◽

P.J. Hooykaas ◽

B. Keller

Keyword(s):

Leaf Development ◽

Leaf Blade ◽

Proximal Part ◽

Dna Binding Domain ◽

Wild Type ◽

Leaf Petiole ◽

Right Border ◽

Lep Gene ◽

The Right ◽

Marginal Meristem

In a screen for leaf developmental mutants we have isolated an activator T-DNA-tagged mutant that produces leaves without a petiole. In addition to that leafy petiole phenotype this lettuce (let) mutant shows aberrant inflorescence branching and silique shape. The LEAFY PETIOLE (LEP) gene is located close to the right border of the T-DNA insert linked with these dominant phenotypes and encodes a protein with a domain with similarity to the DNA binding domain of members of the AP2/EREBP family of transcription factors. Introduction of the activation-tagged LEP gene in wild-type plants conferred all the phenotypic aberrations mentioned above. The leafy petiole phenotype consists of a conversion of the proximal part of the leaf from petiole into leaf blade, which means that leaf development in let is disturbed along the proximodistal axis. Therefore, LEP is involved in either cell division activity in the marginal meristem or patterning along the proximodistal axis.

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Mutations affecting the formation of the notochord in the zebrafish, Danio rerio

Development ◽

10.1242/dev.123.1.103 ◽

1996 ◽

Vol 123 (1) ◽

pp. 103-115 ◽

Cited By ~ 38

Author(s):

J. Odenthal ◽

P. Haffter ◽

E. Vogelsang ◽

M. Brand ◽

F.J. van Eeden ◽

...

Keyword(s):

Sonic Hedgehog ◽

Large Scale ◽

Floor Plate ◽

Precursor Cells ◽

Wild Type ◽

Plate Formation ◽

Notochord Cells ◽

The Right ◽

Adaxial Cells

In a large scale screen for mutants with defects in the embryonic development of the zebrafish we identified mutations in four genes, floating head (flh), momo (mom), no tail (ntl), and doc, that are required for early notochord formation. Mutations in flh and ntl have been described previously, while mom and doc are newly identified genes. Mutant mom embryos lack a notochord in the trunk, and trunk somites from the right and left side of the embryo fuse underneath the neural tube. In this respect mom appears similar to flh. In contrast, notochord precursor cells are present in both ntl and doc embryos. In order to gain a greater understanding of the phenotypes, we have analysed the expression of several axial mesoderm markers in mutant embryos of all four genes. In flh and mom, Ntl expression is normal in the germ ring and tailbud, while the expression of Ntl and other notochord markers in the axial mesodermal region is disrupted. Ntl expression is normal in doc embryos until early somitic stages, when there is a reduction in expression which is first seen in anterior regions of the embryo. This suggests a function for doc in the maintenance of ntl expression. Other notochord markers such as twist, sonic hedgehog and axial are not expressed in the axial mesoderm of ntl embryos, their expression parallels the expression of ntl in the axial mesoderm of mutant doc, flh and mom embryos, indicating that ntl is required for the expression of these markers. The role of doc in the expression of the notochord markers appears indirect via ntl. Floor plate formation is disrupted in most regions in flh and mom mutant embryos but is present in mutant ntl and doc embryos. In mutant embryos with strong ntl alleles the band of cells expressing floor plate markers is broadened. A similar broadening is also observed in the axial mesoderm underlying the floor plate of ntl embryos, suggesting a direct involvement of the notochord precursor cells in floor plate induction. Mutations in all of these four genes result in embryos lacking a horizontal myoseptum and muscle pioneer cells, both of which are thought to be induced by the notochord. These somite defects can be traced back to an impairment of the specification of the adaxial cells during early stages of development. Transplantation of wild-type cells into mutant doc embryos reveals that wild-type notochord cells are sufficient to induce horizontal myoseptum formation in the flanking mutant tissue. Thus doc, like flh and ntl, acts cell autonomously in the notochord. In addition to the four mutants with defects in early notochord formation, we have isolated 84 mutants, defining at least 15 genes, with defects in later stages of notochord development. These are listed in an appendix to this study.

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