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Eric T Kool - One of the best experts on this subject based on the ideXlab platform.
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Efficient isothermal expansion of human telomeric and Minisatellite Repeats by Thermococcus litoralis DNA polymerase
Nucleic acids research, 2005Co-Authors: Jorg S Hartig, Eric T KoolAbstract:Repeating DNA sequences, such as telomeres, centromeres, and micro- and mini-satellites, comprise 50% of the genome and play important roles in regulatory and pathogenic mechanisms. In order to study structures and functions of such repeating sequences, it is important to have simple and efficient methods for making them in vitro. Here, we describe the efficient and convenient expansion of repetitive telomeric and Minisatellite DNA sequences starting from small synthetic templates to final product lengths of several hundreds to thousands of nucleotides by the thermostable DNA polymerase from Thermococcus litoralis (Vent DNA polymerase). This enzyme was so far unknown to catalyze repeat expansion. Either single-stranded or double-stranded DNAs could be produced, depending on nucleotides present. Compared to earlier results obtained with other enzymes, the expansion reaction is highly efficient both in its yield and product length, and proceeds without thermal cycling. Moreover, the products are characterized by a narrow length distribution.
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efficient isothermal expansion of human telomeric and Minisatellite Repeats by thermococcus litoralis dna polymerase
Nucleic Acids Research, 2005Co-Authors: Jorg S Hartig, Eric T KoolAbstract:Repeating DNA sequences, such as telomeres, centromeres, and micro- and mini-satellites, comprise 50% of the genome and play important roles in regulatory and pathogenic mechanisms. In order to study structures and functions of such repeating sequences, it is important to have simple and efficient methods for making them in vitro. Here, we describe the efficient and convenient expansion of repetitive telomeric and Minisatellite DNA sequences starting from small synthetic templates to final product lengths of several hundreds to thousands of nucleotides by the thermostable DNA polymerase from Thermococcus litoralis (Vent DNA polymerase). This enzyme was so far unknown to catalyze repeat expansion. Either single-stranded or double-stranded DNAs could be produced, depending on nucleotides present. Compared to earlier results obtained with other enzymes, the expansion reaction is highly efficient both in its yield and product length, and proceeds without thermal cycling. Moreover, the products are characterized by a narrow length distribution.
Jorg S Hartig - One of the best experts on this subject based on the ideXlab platform.
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Efficient isothermal expansion of human telomeric and Minisatellite Repeats by Thermococcus litoralis DNA polymerase
Nucleic acids research, 2005Co-Authors: Jorg S Hartig, Eric T KoolAbstract:Repeating DNA sequences, such as telomeres, centromeres, and micro- and mini-satellites, comprise 50% of the genome and play important roles in regulatory and pathogenic mechanisms. In order to study structures and functions of such repeating sequences, it is important to have simple and efficient methods for making them in vitro. Here, we describe the efficient and convenient expansion of repetitive telomeric and Minisatellite DNA sequences starting from small synthetic templates to final product lengths of several hundreds to thousands of nucleotides by the thermostable DNA polymerase from Thermococcus litoralis (Vent DNA polymerase). This enzyme was so far unknown to catalyze repeat expansion. Either single-stranded or double-stranded DNAs could be produced, depending on nucleotides present. Compared to earlier results obtained with other enzymes, the expansion reaction is highly efficient both in its yield and product length, and proceeds without thermal cycling. Moreover, the products are characterized by a narrow length distribution.
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efficient isothermal expansion of human telomeric and Minisatellite Repeats by thermococcus litoralis dna polymerase
Nucleic Acids Research, 2005Co-Authors: Jorg S Hartig, Eric T KoolAbstract:Repeating DNA sequences, such as telomeres, centromeres, and micro- and mini-satellites, comprise 50% of the genome and play important roles in regulatory and pathogenic mechanisms. In order to study structures and functions of such repeating sequences, it is important to have simple and efficient methods for making them in vitro. Here, we describe the efficient and convenient expansion of repetitive telomeric and Minisatellite DNA sequences starting from small synthetic templates to final product lengths of several hundreds to thousands of nucleotides by the thermostable DNA polymerase from Thermococcus litoralis (Vent DNA polymerase). This enzyme was so far unknown to catalyze repeat expansion. Either single-stranded or double-stranded DNAs could be produced, depending on nucleotides present. Compared to earlier results obtained with other enzymes, the expansion reaction is highly efficient both in its yield and product length, and proceeds without thermal cycling. Moreover, the products are characterized by a narrow length distribution.
Bisht Prakhar - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms modulating transcribed chromatin topology and suppressing replication fork instability
'Ediciones Universidad de Salamanca', 2017Co-Authors: Bisht PrakharAbstract:[EN]: DNA replication is a risky process for the cell. Chromosomal DNA synthesis takes place at specialized structures (replication forks), which present intrinsic fragility and are preferred substrates of unscheduled recombination events. When replication fork progression is hampered, they can collapse priming DNA breaks and genomic rearrangements. Collision of replication forks with actively transcribed genes is a potential main determinant of replication fork stalling and a number of pathways have been involved in transcribed genomic regions protection. In budding yeast, DNA topoisomerase II (Top2) has been proposed to organize architectural domains to prevent DNA breaks following transcribed regions replication. Factors acting in the interface between transcription and mRNA export, like the THO/TREX complex, also suppress the accumulation of DNA:RNA hybrids forming at negatively supercoiled segments behind transcription bubble. DNA:RNA hybrids, when engaged by replication forks, prime aberrant recombination events and chromosomal abnormalities. Over 26 years ago, a topoisomerase-like catalytic active site was described in the Hpr1 THO component, raising the possibility that the complex modulates transcribed regions topology. To address this point, we mutagenized Hpr1 topoisomerase-like catalytic tyrosine to generate an hpr1-Y590A mutant allele. We found that hpr1-Y590A cells are sensitive to treatment with the replication inhibitor hydroxyurea, suggesting that Hpr1 might play a topological role in promoting replication fork stability. The hpr1-Y590A allele shows synthetic growth defects in combination with top2 mutations, and hpr1-Y590A top2-1 cells accumulate checkpoint activation signals following S-phase completion. This evidence suggests a degree of coordination between Hpr1 and Top2 in resolving topological stress and preventing chromosomal breaks at sites of replication transcription interference. Moreover, putative Hpr1 catalytic tyrosine mutation leads to the accumulation of negatively supercoiled topoisomers in Top1 compromised cells. We propose that the topoisomerase like catalytic tyrosine residue of Hpr1 contributes to modulate DNA topology at transcribed chromatin thus alleviating deleterious collisions between DNA replication and gene transcription machineries. Formation of secondary structures on thetemplate DNA can also destabilize replication forks and prime chromosome rearrangements. Human Minisatellite Repeats, which show highly dynamic changes in repeat number, are prone to form G-quadruplexes, a four-stranded structure in which guanines within a single stranded DNA stretch pair and stack. Using budding yeast model system we have studied the replication of the human sub-telomeric Minisatellites CEB25 and c-MYC. By 2D gel electrophoresis, we found that aberrant replication intermediates accumulate at CEB25 harboring regions in cells lacking Pif1 helicase. Likewise during the replication of c-MYC human Minisatellite, we observed the accumulation of recombination intermediates in cells in which Pif1 activity is compromised. These defects observed are consistent with the proposed role of Pif1 in unwinding G-quadruplexes. We propose that these replication defects upon G-quadruplex stabilization in Pif1-ablated cells Repeats reflect strand discontinuities in CEB25 Minisatellite, while they determine fork breakage and unscheduled recombination repair attempts within the c-MYC Minisatellite.[ES]: La replicación del ADN es un proceso peligroso para la célula. La síntesis del ADN cromosómico tiene lugar en las estructuras especializadas (horquillas de replicación), las cuales presentan una fragilidad intrínseca y son sustrato frequente de eventos de recombinación no programados. Cuando se impide la progresión de las horquillas de replicación, pueden llegar colapsar y originar roturas del ADN y reordenamientos genómicos. La colisión de horquillas de replicación con genes activamente transcritos es un importante determinante potencial del bloqueo de las horquillas de replicación y un gran número de rutas celulares se ven involucradas en la protección de regiones genómicas transcritas. En la levadura en gemación, se ha propuesto que la topoisomerasa de ADN II (Top2) organiza dominios arquitectónicos para prevenir las roturas de ADN derivadas de la replicación de regiones transcritas. Algunos factores que actúan en la conjunción entre la transcripción y laexportación de ARNm, como el complejo THO/TREX, también previenen la acumulación de híbridos de ADN y ARN que se forman en segmentos superenrollados negativamente detrás de la burbuja de transcripción. Cuando las horquillas de replicación encuentran híbridos de ADN y ARN se originan eventos de recombinación aberrantes y anomalías cromosómicas. Hace unos 26 años, se describió un sitio activo catalítico similar a la topoisomerasa en el componente Hpr1 del complejo THO, planteando la posibilidad de que este complejo modulase la topología de las regiones transcritas. Con esto en mente, mutamos la tirosina catalítica similar a la de topoisomerasa para generar un alelo mutante hpr1-Y590A. Descubrimos que los células hpr1-Y590A son sensibles al tratamiento con el inhibidor de replicación hidroxiurea, lo que sugiere que el Hpr1 puede desempeñar un papel topológico a la hora de fomentar la estabilidad de las horquillas de replicación. El alelo hpr1-Y590A muestra defectos de crecimiento sintético en combinación con mutaciones top2, y las células hpr1-Y590A top2-1 acumulan señales de activación de puntos de control tras la finalización de la fase S. Esta evidencia sugiere un grado de coordinación entre Hpr1 y Top2 a la hora de aliviar estrés topológico y prevenir roturas cromosómicas en los lugares donde interfieren replicación y transcripción. Además, la tirosina catalítica putativa de Hpr1 conduce a la acumulación de topoisomeros superenrollados negativamente en células comprometidas en la actividad de Top1. Proponemos que el residuo de la tirosina catalítica topoisomerase similara la de topoisomerasa de Hpr1 contribuye a la modulación de la topología del ADN en la cromatina transcrita, paliando así colisiones dañinas entre las maquinarias de replicación del ADN y de transcripción génica. La formación de estructuras secundarias en el molde de ADN puede desestabilizar a las horquillas de replicación y dar lugar a reordenamientos de cromosomas. Las repeticiones de minisatélites humanos, que muestran cambios muy dinámicos en el número de repeticiones, tienden a formar quadruplexes de Gs, estructuras de cuatro cadenas en las que las guaninas de un tramo de ADN monocatenario se emparejan y se apilan. Mediante el sistema de levadura en gemación, hemos estudiado la replicación de los minisatélites subteloméricos humanos CEB25 y c-MYC. A través de electroforesis en gel de 2D observamos que los intermediarios replicativos aberrantes se acumulan en las regiones donde se ubica CEB25 en células carente de la helicasa Pif1. Del mismo modo, durante 3 la replicación del minisatélite humano c-MYC, observamos acumulaciones de putativos intermediarios de recombinación en células en las que la actividad Pif1 se encuentra comprometida. Estos defectos son coherentes con la función propuesta de Pif1 a la hora de desenrollar quadruplexes de Gs. Proponemos que estos defectos de replicación en la estabilización de quadruplexes de Gs en repeticiones de células con ablación de Pif1 reflejan discontinuidades de cadena naciente en el minisatélite CEB25, mientras determinan roturas de horquillas e intentos no programados de reparación de mediante recombinación en el minisatélite c-MYC.Peer Reviewe
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Mechanisms modulating transcribed chromatin topology and suppressing replication fork instability
2017Co-Authors: Bisht PrakharAbstract:[ES] La replicación del ADN es un proceso peligroso para la célula. La síntesis del ADN cromosómico tiene lugar en las estructuras especializadas (horquillas de replicación), las cuales presentan una fragilidad intrínseca y son sustrato frequente de eventos de recombinación no programados. Cuando se impide la progresión de las horquillas de replicación, pueden llegar colapsar y originar roturas del ADN y reordenamientos genómicos. La colisión de horquillas de replicación con genes activamente transcritos es un importante determinante potencial del bloqueo de las horquillas de replicación y un gran número de rutas celulares se ven involucradas en la protección de regiones genómicas transcritas. En la levadura en gemación, se ha propuesto que la topoisomerasa de ADN II (Top2) organiza dominios arquitectónicos para prevenir las roturas de ADN derivadas de la replicación de regiones transcritas. Algunos factores que actúan en la conjunción entre la transcripción y la exportación de ARNm, como el complejo THO/TREX, también previenen la acumulación de híbridos de ADN y ARN que se forman en segmentos superenrollados negativamente detrás de la burbuja de transcripción. Cuando las horquillas de replicación encuentran híbridos de ADN y ARN se originan eventos de recombinación aberrantes y anomalías cromosómicas. Hace unos 26 años, se describió un sitio activo catalítico similar a la topoisomerasa en el componente Hpr1 del complejo THO, planteando la posibilidad de que este complejo modulase la topología de las regiones transcritas. Con esto en mente, mutamos la tirosina catalítica similar a la de topoisomerasa para generar un alelo mutante hpr1-Y590A. Descubrimos que los células hpr1-Y590A son sensibles al tratamiento con el inhibidor de replicación hidroxiurea, lo que sugiere que el Hpr1 puede desempeñar un papel topológico a la hora de fomentar la estabilidad de las horquillas de replicación. El alelo hpr1-Y590A muestra defectos de crecimiento sintético en combinación con mutaciones top2, y las células hpr1-Y590A top2-1 acumulan señales de activación de puntos de control tras la finalización de la fase S. Esta evidencia sugiere un grado de coordinación entre Hpr1 y Top2 a la hora de aliviar estrés topológico y prevenir roturas cromosómicas en los lugares donde interfieren replicación y transcripción. Además, la tirosina catalítica putativa de Hpr1 conduce a la acumulación de topoisomeros superenrollados negativamente en células comprometidas en la actividad de Top1. Proponemos que el residuo de la tirosina catalítica topoisomerase similar a la de topoisomerasa de Hpr1 contribuye a la modulación de la topología del ADN en la cromatina transcrita, paliando así colisiones dañinas entre las maquinarias de replicación del ADN y de transcripción génica. La formación de estructuras secundarias en el molde de ADN puede desestabilizar a las horquillas de replicación y dar lugar a reordenamientos de cromosomas. Las repeticiones de minisatélites humanos, que muestran cambios muy dinámicos en el número de repeticiones, tienden a formar quadruplexes de Gs, estructuras de cuatro cadenas en las que las guaninas de un tramo de ADN monocatenario se emparejan y se apilan. Mediante el sistema de levadura en gemación, hemos estudiado la replicación de los minisatélites subteloméricos humanos CEB25 y c-MYC. A través de electroforesis en gel de 2D observamos que los intermediarios replicativos aberrantes se acumulan en las regiones donde se ubica CEB25 en células carente de la helicasa Pif1. Del mismo modo, durante 3 la replicación del minisatélite humano c-MYC, observamos acumulaciones de putativos intermediarios de recombinación en células en las que la actividad Pif1 se encuentra comprometida. Estos defectos son coherentes con la función propuesta de Pif1 a la hora de desenrollar quadruplexes de Gs. Proponemos que estos defectos de replicación en la estabilización de quadruplexes de Gs en repeticiones de células con ablación de Pif1 reflejan discontinuidades de cadena naciente en el minisatélite CEB25, mientras determinan roturas de horquillas e intentos no programados de reparación de mediante recombinación en el minisatélite c-MYC HIPÓTESIS DE TRABAJO Con los datos obtenidos, formulamos la hipótesis de que la subunidad Hpr1 del complejo THO presenta una actividad equivalente a topoisomerasa para aliviar el estrés torsional acumulado detrás de la maquinaria transcripcional, que de no concluir favorece la formación de híbridos ARN:ADN, a los que se les atribuye el bloqueo de la replicación de horquillas. De la misma manera, postulamos también que se requiere específicamente la helicasa Pif1 para determinar las estructuras quadruplexes de Gs formados por los minisatélites humanos CEB25 y c-MYC. OBJETIVOS GENERALES El objetivo principal de esta tesis es investigar los mecanismos que impiden la inestabilidad de las horquillas de replicación en los contextos de 1) un conflicto entre las maquinarias de replicación con la transcripción génica activa y 2) durante la progresión de replicación a lo largo de estructuras secundarias del ADN formando quadruplexes de Gs. Los principales objetivos son: 1) definir el papel de la subunidad Hpr1 del complejo THO a la hora de modular la topología de la cromatina transcrita y de proteger la integridad de la replicación de horquillas; 2) estudiar la contribución de la helicasa Pif1,esencial para la replicación de diferentes quadruplexes de Gs y la formación de secuencias de minisatélites humanos. CONCLUSIONES GENERALES Teniendo en cuenta los datos presentados en este estudio, concluimos lo siguiente: I) un residuo de tirosina catalítica similar a la de topoisomerasa está bien conservada en ortólogos Hpr1 desde levaduras hasta eucariotas superiores. II) el residuo catalítico topoisomerase-like de Hpr1 es necesario para que las células protejan las horquillas de replicación en condiciones de estrés replicativo. III) Hpr1 y Top2 probablemente comparten una función topológica solapante a la hora de favorecer el crecimiento celular y de mantener la estabilidad de horquillas bloqueadas. IV) Es posible que el residuo catalítico similar a topo Hpr1 coopere con Top2 a la hora de evitar conflictos entre la replicación y la transcripción para proteger la integridad cromosómica. V) el residuo de tirosina catalítica de Hpr1 podría actuar como topoisomerasa para aliviar estrés torsional acumulado detrás de la maquinaria ARN polimerasa II durante la transcripción génica. VI) la replicación del minisatélite humano CEB25 en ausencia de la helicasa Pif1 probablemente de lugar a discontinuidades en las cadenas nacientes dentro de la secuencia de repetición. VII) la replicación del minisatélite humano c-MYC en células con ablación de la actividad Pif1 conduce a la acumulación de intermediarios consistentes con intentos de reparación de recombinación homólogos de las horquillas de replicación colapsadas.[EN] DNA replication is a risky process for the cell. Chromosomal DNA synthesis takes place at specialized structures (replication forks), which present intrinsic fragility and are preferred substrates of unscheduled recombination events. When replication fork progression is hampered, they can collapse priming DNA breaks and genomic rearrangements. Collision of replication forks with actively transcribed genes is a potential main determinant of replication fork stalling and a number of pathways have been involved in transcribed genomic regions protection. In budding yeast, DNA topoisomerase II (Top2) has been proposed to organize architectural domains to prevent DNA breaks following transcribed regions replication. Factors acting in the interface between transcription and mRNA export, like the THO/TREX complex, also suppress the accumulation of DNA:RNA hybrids forming at negatively supercoiled segments behind transcription bubble. DNA:RNA hybrids, when engaged by replication forks, prime aberrant recombination events and chromosomal abnormalities. Over 26 years ago, a topoisomerase-like catalytic active site was described in the Hpr1 THO component, raising the possibility that the complex modulates transcribed regions topology. To address this point, we mutagenized Hpr1 topoisomerase-like catalytic tyrosine to generate an hpr1-Y590A mutant allele. We found that hpr1-Y590A cells are sensitive to treatment with the replication inhibitor hydroxyurea, suggesting that Hpr1 might play a topological role in promoting replication fork stability. The hpr1-Y590A allele shows synthetic growth defects in combination with top2 mutations, and hpr1-Y590A top2-1 cells accumulate checkpoint activation signals following S-phase completion. This evidence suggests a degree of coordination between Hpr1 and Top2 in resolving topological stress and preventing chromosomal breaks at sites of replication transcription interference. Moreover, putative Hpr1 catalytic tyrosine mutation leads to the accumulation of negatively supercoiled topoisomers in Top1 compromised cells. We propose that the topoisomerase like catalytic tyrosine residue of Hpr1 contributes to modulate DNA topology at transcribed chromatin thus alleviating deleterious collisions between DNA replication and gene transcription machineries. Formation of secondary structures on the template DNA can also destabilize replication forks and prime chromosome rearrangements. Human Minisatellite Repeats, which show highly dynamic changes in repeat number, are prone to form G-quadruplexes, a four-stranded structure in which guanines within a single stranded DNA stretch pair and stack. Using budding yeast model system we have studied the replication of the human sub-telomeric Minisatellites CEB25 and c-MYC. By 2D gel electrophoresis, we found that aberrant replication intermediates accumulate at CEB25 harboring regions in cells lacking Pif1 helicase. Likewise during the replication of c-MYC human Minisatellite, we observed the accumulation of recombination intermediates in cells in which Pif1 activity is compromised. These defects observed are consistent with the proposed role of Pif1 in unwinding G-quadruplexes. We propose that these replication defects upon G-quadruplex stabilization in Pif1-ablated cells Repeats reflect strand discontinuities in CEB25 Minisatellite, while they determine fork breakage and unscheduled recombination repair attempts within the c-MYC Minisatellite
Rainer Blasczyk - One of the best experts on this subject based on the ideXlab platform.
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weak blood group b phenotypes may be caused by variations in the ccaat binding factor nf y enhancer region of the abo gene
Transfusion, 2007Co-Authors: Axel Seltsam, Franz F. Wagner, Daniela Grüger, Christa Das Gupta, Christina Badedoeding, Rainer BlasczykAbstract:BACKGROUND: Binding of CCAAT-binding factor NF-Y (CBF/NF-Y) to a 43-bp repeat unit in the Minisatellite region in the 5′ region of the ABO gene (CBF/NF-Y enhancer region) plays an important role in regulating the transcription of ABO genes. The common ABO alleles were found to have CBF/NF-Y enhancer regions with specific numbers of 43-bp Minisatellite Repeats. MATERIAL AND METHODS: Blood samples from four healthy blood donors with weak B phenotypes were subjected to extensive ABO genotyping, including nucleotide sequencing of the 5′ regulatory region containing the CBF/NF-Y enhancer. RESULTS: The coding region of the ABO genes exhibited common ABO*B101-heterozygous genotypes in all samples, but unexpected variations were observed in the CBF/NF-Y enhancer region. In two cases, the CBF/NF-Y enhancer motifs did not exhibit the expected ABO allele dependency. One, an ABweak sample was heterozygous for ABO*A101 and ABO*B101 but homozygous for the ABO*B101-specific CBF/NF-Y motif. The second had a common ABO*B101/ABO*O01 genotype but was heterozygous for ABO*A101- and ABO*O01-specific enhancer motifs. In the other two samples, novel CBF/NF-Y motifs were found. One contained a shortened version of an otherwise ABO*B101-specific CBF/NF-Y motif, and the other had a single-base substitution located 12 bp upstream from the beginning of the first 43-bp repeat of an ABO*B101-specific CBF/NF-Y enhancer sequence. CONCLUSION: The frequency of variations in the CBF/NF-Y region of the ABO gene in these samples with presumably common ABO*B101 alleles suggests that weak blood group B phenotypes may be caused by sequence variations in the CBF/NF-Y regulatory region.
Axel Seltsam - One of the best experts on this subject based on the ideXlab platform.
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weak blood group b phenotypes may be caused by variations in the ccaat binding factor nf y enhancer region of the abo gene
Transfusion, 2007Co-Authors: Axel Seltsam, Franz F. Wagner, Daniela Grüger, Christa Das Gupta, Christina Badedoeding, Rainer BlasczykAbstract:BACKGROUND: Binding of CCAAT-binding factor NF-Y (CBF/NF-Y) to a 43-bp repeat unit in the Minisatellite region in the 5′ region of the ABO gene (CBF/NF-Y enhancer region) plays an important role in regulating the transcription of ABO genes. The common ABO alleles were found to have CBF/NF-Y enhancer regions with specific numbers of 43-bp Minisatellite Repeats. MATERIAL AND METHODS: Blood samples from four healthy blood donors with weak B phenotypes were subjected to extensive ABO genotyping, including nucleotide sequencing of the 5′ regulatory region containing the CBF/NF-Y enhancer. RESULTS: The coding region of the ABO genes exhibited common ABO*B101-heterozygous genotypes in all samples, but unexpected variations were observed in the CBF/NF-Y enhancer region. In two cases, the CBF/NF-Y enhancer motifs did not exhibit the expected ABO allele dependency. One, an ABweak sample was heterozygous for ABO*A101 and ABO*B101 but homozygous for the ABO*B101-specific CBF/NF-Y motif. The second had a common ABO*B101/ABO*O01 genotype but was heterozygous for ABO*A101- and ABO*O01-specific enhancer motifs. In the other two samples, novel CBF/NF-Y motifs were found. One contained a shortened version of an otherwise ABO*B101-specific CBF/NF-Y motif, and the other had a single-base substitution located 12 bp upstream from the beginning of the first 43-bp repeat of an ABO*B101-specific CBF/NF-Y enhancer sequence. CONCLUSION: The frequency of variations in the CBF/NF-Y region of the ABO gene in these samples with presumably common ABO*B101 alleles suggests that weak blood group B phenotypes may be caused by sequence variations in the CBF/NF-Y regulatory region.