Zbigniew Przybecki - Academia.edu (original) (raw)

Papers by Zbigniew Przybecki

Photonics Applications in Astronomy, Communications, Industry, and High-Energy Physics Experiments 2014, 2014

Photonics Applications in Astronomy, Communications, Industry, and High-Energy Physics Experiments 2014, 2014

Cellular & Molecular Biology Letters

This study describes an effective method of in situ RT-PCR (RT-ISPCR) that was developed to local... more This study describes an effective method of in situ RT-PCR (RT-ISPCR) that was developed to localize gene expression in plant tissues. This RT-PCR technique was performed on sectioned tissues of female buds of the cucumber GY3 inbred line. The CUS1 gene, encoding the MADS-box type (agamus-like) protein, the expression pattern of which was described earlier , was used as a marker gene for optimisation of steps in the in situ RT-PCR inside the cells. For the identification of RT-PCR products inside the cells of the female buds, they were fixed in FAA solution, embedded in Paraplast Plus and cut into 7µm thick sections which were dewaxed by immersion in HistoClear and dehydrated with ethanol. They were washed in water, then in 0.02M HCl, 2xSSC and PBS buffer. In the next step of tissue pretreatment, the sections were digested with 1% pectinase. As shown, the pectinase treatment proved to be a crucial step in the tissue preparation procedure to get successful RT-PCR products. After washing in PBS buffer, the sections were digested with protease K followed by incubation with RNase-free DNase I, and subsequently washed in 2xSSC, 1xSSC and 0.5xSSC and finally in DEPC-treated water. Then the sections were covered with 50µl of the RT-PCR reaction mixture supplemented with 0.5µM digoxigenin dUTP and sealed with a coverslip. After amplification in situ the PCR products were identified with anti-digoxigenin antibody (Roche Molecular Biochemicals), conjugated with alkaline phosphatase. The data obtained showed that specific signals reflecting CUS1 gene expression were detected in the female flower buds of cucumber. The specificity of the in situ RT-PCR protocol was confirmed by dot blot hybridization of RT-PCR products with CUS1 cDNA probe.

Cytogenetic and Genome Research, 2014

Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential g... more Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential genetic resource for Cucumis crops. Although some cytogenetic research has been reported, there is no study of karyotyping in this species. Here, we used 4',6-diamidino-2-phenylindole and chromomycin A3 staining to identify 12 pairs of chromosomes in early-metaphase cells. Fluorescence in situ hybridization revealed the chromosomal distribution patterns of the 5S and 45S ribosomal DNA (rDNA) genes, telomeres, and 3 different satellite repeats. The 2 major signals of the 45S rDNA were located on the satellite of chromosome 11, and the 2 signals of the 5S rDNA and 2 minor signals of the 45S rDNA were located on chromosome 12. The telomere probes hybridized to the ends of all chromosomes. The 3 satellite DNAs were localized at the ends of chromosomes 1, 2, 4-10, and at the end of the short arm of chromosome 3. In summary, we reported the identification of all chromosomes of C. metuliferus. We also depicted the location of 5S and 45S rDNA, the telomere motif sequence, CmetSat1, CmetSatT2, and CmetmSat1 in an ideogram. © 2014 S. Karger AG, Basel.

Nature Precedings, 2011

... (Cucumis sativus L. cv. Borszczagovski) genome – the most efficient way to begin post genomic... more ... (Cucumis sativus L. cv. Borszczagovski) genome – the most efficient way to begin post genomic era Woycicki R., Witkowicz J., Pawelkowicz M, Siedlecka E., Gutman W., Plader W., Seroczynska A., Smiech M., Niemirowicz-Szczytt K., Karpinski S, Malepszy S., Przybecki Z. Dept. ...

Cellular & molecular biology letters, 2004

Isolations of polymorphic sequences of two pairs of the NIL lines of cucumber (Cucumis sativus L.... more Isolations of polymorphic sequences of two pairs of the NIL lines of cucumber (Cucumis sativus L.), which differ with respect to sex, were carried out using the subtraction hybridization methods of DSC (Differential Subtraction Chain) and GDDSC (Genetically Directed DSC). 266 DSC tags were isolated from the entire genome region, and 38 GDDSC tags were isolated from the region containing the sex genes. Based on the obtained results, the methods used may be considered highly effective. The attained sequences, like 11 AFLP clones obtained earlier [Witkowicz, J. et al. Cell. Mol. Biol. Lett. 8 (2003) 375-381], were characterized by analyzing their hybridization with differential (dhaom) and subtractive cDNA libraries (cDNAsubtractom) from 1- to 2- mm floral buds of the same lines, and by the sequencing of 28 tags. A high average degree of homology was found to exist in the genpolom to dhom and cDNAsubtractom, particularly in the case of "dominant" (when the tester used was a l...

Photonics Applications in Astronomy, Communications, Industry, and High-Energy Physics Experiments 2014, 2014

PRINS and In Situ PCR Protocols, 2006

In situ detection techniques allow specific nucleic acid sequences to be exposed in morphological... more In situ detection techniques allow specific nucleic acid sequences to be exposed in morphologically preserved tissue sections. In combination with immunocytochemistry, in situ detection can relate microscopic topological information to gene activity at the transcript or protein levels in specific tissues. The advantage of in situ methods over the conventional techniques (e.g., Northern blot, reverse transcription polymerase chain reaction [RT-PCR], or real-time PCR) is that they allow the investigatation of the putative spatial distribution of nucleic acid products activity in a heterogeneous cell population. In this chapter, we describe a protocol for in situ RT-PCR detection of specific messenger RNA in cucumber (Cucumis sativus), although this protocol can be used for any plant species, floral buds, and somatic embryo tissue sections on glass microscope slides. A successful in situ RT-PCR procedure requires the optimization of many conditions related to the tissue types used, for example, a cell's age, size, and composition, which may influence the detection of RT-PCR products, as well as specific transcript availability. Moreover, parameters, such as the fixation time, thermal cycling set-up, and the time of detection of RT-PCR products, also should be optimized. The importance of the other factors also is estimated in the protocol. In addition several types of controls that are necessary for a trustworthy in situ RT-PCR method are being discussed.

PLoS ONE, 2011

Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas an... more Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas and secondary domestication regions includes highly divergent climate conditions e.g. temperate and subtropical. We wanted to uncover adaptive genome differences between the cucumber cultivars and what sort of evolutionary molecular mechanisms regulate genetic adaptation of plants to different ecosystems and organism biodiversity. Here we present the draft genome sequence of the Cucumis sativus genome of the North-European Borszczagowski cultivar (line B10) and comparative genomics studies with the known genomes of: C. sativus (Chinese cultivar -Chinese Long (line 9930)), Arabidopsis thaliana, Populus trichocarpa and Oryza sativa. Cucumber genomes show extensive chromosomal rearrangements, distinct differences in quantity of the particular genes (e.g. involved in photosynthesis, respiration, sugar metabolism, chlorophyll degradation, regulation of gene expression, photooxidative stress tolerance, higher non-optimal temperatures tolerance and ammonium ion assimilation) as well as in distributions of abscisic acid-, dehydration-and ethylene-responsive cisregulatory elements (CREs) in promoters of orthologous group of genes, which lead to the specific adaptation features. Abscisic acid treatment of non-acclimated Arabidopsis and C. sativus seedlings induced moderate freezing tolerance in Arabidopsis but not in C. sativus. This experiment together with analysis of abscisic acid-specific CRE distributions give a clue why C. sativus is much more susceptible to moderate freezing stresses than A. thaliana. Comparative analysis of all the five genomes showed that, each species and/or cultivars has a specific profile of CRE content in promoters of orthologous genes. Our results constitute the substantial and original resource for the basic and applied research on environmental adaptations of plants, which could facilitate creation of new crops with improved growth and yield in divergent conditions.

Cellular & Molecular Biology Letters, 2008

Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, w... more Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, which are used for the physical mapping, identification and isolation of genes, and for gene sequencing. A BAC genomic library was constructed from high molecular weight DNA (HMW DNA) obtained from nuclei of the cucumber (Cucumis sativus L. cv. Borszczagowski; B10 line). The DNA was digested with the HindIII restriction enzyme and ligated into the pCC1BAC vector. The library consists of 34,560 BAC clones with an average insert size of 135 kb, and 12.7x genome coverage. Screening the library for chloroplast and mitochondrial DNA content indicated an exceptionally low 0.26% contamination with chloroplast DNA and 0.3% with mitochondrial DNA.

Acta Physiologiae Plantarum, 2001

... Ewa Urbahczyk-Wochniak, Zbigniew Przybecki ... 1991b). The homeotic genes AGA-MOUS and APETAL... more ... Ewa Urbahczyk-Wochniak, Zbigniew Przybecki ... 1991b). The homeotic genes AGA-MOUS and APETALA2 appear to serve cadastral role in determining each other's pattern of expres-sion in addition to their organ specification activi-ties. ...

Cellular & molecular biology letters, 2003

In this study, we found flower cDNA clones which may be connected with the development of flower ... more In this study, we found flower cDNA clones which may be connected with the development of flower sex in cucumber. Two pairs of nearly-isogenic lines: gynoecious GY3 (FFMMGG) versus hermaphrodite HGY3 (FFmmGG) and monoecious B10 (ffMMGG) versus gynoecious 2gg (ffMMgg) were used for clone isolation. To obtain differentially-expressed clones, we applied the differential screening method. 454 clones from GY3 and 478 from B10 cDNA libraries were isolated. The results of RFLP analysis with 56 cDNA clones showed no clones which cosegregated with sex in cucumber. The 28 cDNA B10 and 33 cDNA GY3 clones isolated using the differential screening method were sequenced. Some of them seem to may play a role in cell differentiation or flower development. Among the 61 identified clones, 14 show high homology to plant proteins, although of unknown function. 11 show high homology to known proteins, and the possible function of some of them is discussed. For 3 clones, no significant similarity was fou...

Photonics Applications in Astronomy, Communications, Industry, and High-Energy Physics Experiments 2014, 2014

Photonics Applications in Astronomy, Communications, Industry, and High-Energy Physics Experiments 2014, 2014

Cellular & Molecular Biology Letters

This study describes an effective method of in situ RT-PCR (RT-ISPCR) that was developed to local... more This study describes an effective method of in situ RT-PCR (RT-ISPCR) that was developed to localize gene expression in plant tissues. This RT-PCR technique was performed on sectioned tissues of female buds of the cucumber GY3 inbred line. The CUS1 gene, encoding the MADS-box type (agamus-like) protein, the expression pattern of which was described earlier , was used as a marker gene for optimisation of steps in the in situ RT-PCR inside the cells. For the identification of RT-PCR products inside the cells of the female buds, they were fixed in FAA solution, embedded in Paraplast Plus and cut into 7µm thick sections which were dewaxed by immersion in HistoClear and dehydrated with ethanol. They were washed in water, then in 0.02M HCl, 2xSSC and PBS buffer. In the next step of tissue pretreatment, the sections were digested with 1% pectinase. As shown, the pectinase treatment proved to be a crucial step in the tissue preparation procedure to get successful RT-PCR products. After washing in PBS buffer, the sections were digested with protease K followed by incubation with RNase-free DNase I, and subsequently washed in 2xSSC, 1xSSC and 0.5xSSC and finally in DEPC-treated water. Then the sections were covered with 50µl of the RT-PCR reaction mixture supplemented with 0.5µM digoxigenin dUTP and sealed with a coverslip. After amplification in situ the PCR products were identified with anti-digoxigenin antibody (Roche Molecular Biochemicals), conjugated with alkaline phosphatase. The data obtained showed that specific signals reflecting CUS1 gene expression were detected in the female flower buds of cucumber. The specificity of the in situ RT-PCR protocol was confirmed by dot blot hybridization of RT-PCR products with CUS1 cDNA probe.

Cytogenetic and Genome Research, 2014

Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential g... more Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential genetic resource for Cucumis crops. Although some cytogenetic research has been reported, there is no study of karyotyping in this species. Here, we used 4',6-diamidino-2-phenylindole and chromomycin A3 staining to identify 12 pairs of chromosomes in early-metaphase cells. Fluorescence in situ hybridization revealed the chromosomal distribution patterns of the 5S and 45S ribosomal DNA (rDNA) genes, telomeres, and 3 different satellite repeats. The 2 major signals of the 45S rDNA were located on the satellite of chromosome 11, and the 2 signals of the 5S rDNA and 2 minor signals of the 45S rDNA were located on chromosome 12. The telomere probes hybridized to the ends of all chromosomes. The 3 satellite DNAs were localized at the ends of chromosomes 1, 2, 4-10, and at the end of the short arm of chromosome 3. In summary, we reported the identification of all chromosomes of C. metuliferus. We also depicted the location of 5S and 45S rDNA, the telomere motif sequence, CmetSat1, CmetSatT2, and CmetmSat1 in an ideogram. © 2014 S. Karger AG, Basel.

Nature Precedings, 2011

... (Cucumis sativus L. cv. Borszczagovski) genome – the most efficient way to begin post genomic... more ... (Cucumis sativus L. cv. Borszczagovski) genome – the most efficient way to begin post genomic era Woycicki R., Witkowicz J., Pawelkowicz M, Siedlecka E., Gutman W., Plader W., Seroczynska A., Smiech M., Niemirowicz-Szczytt K., Karpinski S, Malepszy S., Przybecki Z. Dept. ...

Cellular & molecular biology letters, 2004

Isolations of polymorphic sequences of two pairs of the NIL lines of cucumber (Cucumis sativus L.... more Isolations of polymorphic sequences of two pairs of the NIL lines of cucumber (Cucumis sativus L.), which differ with respect to sex, were carried out using the subtraction hybridization methods of DSC (Differential Subtraction Chain) and GDDSC (Genetically Directed DSC). 266 DSC tags were isolated from the entire genome region, and 38 GDDSC tags were isolated from the region containing the sex genes. Based on the obtained results, the methods used may be considered highly effective. The attained sequences, like 11 AFLP clones obtained earlier [Witkowicz, J. et al. Cell. Mol. Biol. Lett. 8 (2003) 375-381], were characterized by analyzing their hybridization with differential (dhaom) and subtractive cDNA libraries (cDNAsubtractom) from 1- to 2- mm floral buds of the same lines, and by the sequencing of 28 tags. A high average degree of homology was found to exist in the genpolom to dhom and cDNAsubtractom, particularly in the case of "dominant" (when the tester used was a l...

Photonics Applications in Astronomy, Communications, Industry, and High-Energy Physics Experiments 2014, 2014

PRINS and In Situ PCR Protocols, 2006

In situ detection techniques allow specific nucleic acid sequences to be exposed in morphological... more In situ detection techniques allow specific nucleic acid sequences to be exposed in morphologically preserved tissue sections. In combination with immunocytochemistry, in situ detection can relate microscopic topological information to gene activity at the transcript or protein levels in specific tissues. The advantage of in situ methods over the conventional techniques (e.g., Northern blot, reverse transcription polymerase chain reaction [RT-PCR], or real-time PCR) is that they allow the investigatation of the putative spatial distribution of nucleic acid products activity in a heterogeneous cell population. In this chapter, we describe a protocol for in situ RT-PCR detection of specific messenger RNA in cucumber (Cucumis sativus), although this protocol can be used for any plant species, floral buds, and somatic embryo tissue sections on glass microscope slides. A successful in situ RT-PCR procedure requires the optimization of many conditions related to the tissue types used, for example, a cell's age, size, and composition, which may influence the detection of RT-PCR products, as well as specific transcript availability. Moreover, parameters, such as the fixation time, thermal cycling set-up, and the time of detection of RT-PCR products, also should be optimized. The importance of the other factors also is estimated in the protocol. In addition several types of controls that are necessary for a trustworthy in situ RT-PCR method are being discussed.

PLoS ONE, 2011

Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas an... more Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas and secondary domestication regions includes highly divergent climate conditions e.g. temperate and subtropical. We wanted to uncover adaptive genome differences between the cucumber cultivars and what sort of evolutionary molecular mechanisms regulate genetic adaptation of plants to different ecosystems and organism biodiversity. Here we present the draft genome sequence of the Cucumis sativus genome of the North-European Borszczagowski cultivar (line B10) and comparative genomics studies with the known genomes of: C. sativus (Chinese cultivar -Chinese Long (line 9930)), Arabidopsis thaliana, Populus trichocarpa and Oryza sativa. Cucumber genomes show extensive chromosomal rearrangements, distinct differences in quantity of the particular genes (e.g. involved in photosynthesis, respiration, sugar metabolism, chlorophyll degradation, regulation of gene expression, photooxidative stress tolerance, higher non-optimal temperatures tolerance and ammonium ion assimilation) as well as in distributions of abscisic acid-, dehydration-and ethylene-responsive cisregulatory elements (CREs) in promoters of orthologous group of genes, which lead to the specific adaptation features. Abscisic acid treatment of non-acclimated Arabidopsis and C. sativus seedlings induced moderate freezing tolerance in Arabidopsis but not in C. sativus. This experiment together with analysis of abscisic acid-specific CRE distributions give a clue why C. sativus is much more susceptible to moderate freezing stresses than A. thaliana. Comparative analysis of all the five genomes showed that, each species and/or cultivars has a specific profile of CRE content in promoters of orthologous genes. Our results constitute the substantial and original resource for the basic and applied research on environmental adaptations of plants, which could facilitate creation of new crops with improved growth and yield in divergent conditions.

Cellular & Molecular Biology Letters, 2008

Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, w... more Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, which are used for the physical mapping, identification and isolation of genes, and for gene sequencing. A BAC genomic library was constructed from high molecular weight DNA (HMW DNA) obtained from nuclei of the cucumber (Cucumis sativus L. cv. Borszczagowski; B10 line). The DNA was digested with the HindIII restriction enzyme and ligated into the pCC1BAC vector. The library consists of 34,560 BAC clones with an average insert size of 135 kb, and 12.7x genome coverage. Screening the library for chloroplast and mitochondrial DNA content indicated an exceptionally low 0.26% contamination with chloroplast DNA and 0.3% with mitochondrial DNA.

Acta Physiologiae Plantarum, 2001

... Ewa Urbahczyk-Wochniak, Zbigniew Przybecki ... 1991b). The homeotic genes AGA-MOUS and APETAL... more ... Ewa Urbahczyk-Wochniak, Zbigniew Przybecki ... 1991b). The homeotic genes AGA-MOUS and APETALA2 appear to serve cadastral role in determining each other's pattern of expres-sion in addition to their organ specification activi-ties. ...

Cellular & molecular biology letters, 2003

In this study, we found flower cDNA clones which may be connected with the development of flower ... more In this study, we found flower cDNA clones which may be connected with the development of flower sex in cucumber. Two pairs of nearly-isogenic lines: gynoecious GY3 (FFMMGG) versus hermaphrodite HGY3 (FFmmGG) and monoecious B10 (ffMMGG) versus gynoecious 2gg (ffMMgg) were used for clone isolation. To obtain differentially-expressed clones, we applied the differential screening method. 454 clones from GY3 and 478 from B10 cDNA libraries were isolated. The results of RFLP analysis with 56 cDNA clones showed no clones which cosegregated with sex in cucumber. The 28 cDNA B10 and 33 cDNA GY3 clones isolated using the differential screening method were sequenced. Some of them seem to may play a role in cell differentiation or flower development. Among the 61 identified clones, 14 show high homology to plant proteins, although of unknown function. 11 show high homology to known proteins, and the possible function of some of them is discussed. For 3 clones, no significant similarity was fou...