Nuclear genome size and karyotype analysis in Papaver for BAC library construction
The objective of the research carried out is study of the pathway of alkaloid production in Papaver species and cell lines, and integration data on physical mapping of newly developed marker DNA sequences with existing difference in expression of genes for key enzymes of alkaloid biosynthesis. This...
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| Дата: | 2005 |
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Nuclear genome size and karyotype analysis in Papaver for BAC library construction / T.K. Kyrylenko, O.I. Martynenko, O.G. Alkhimova // Биополимеры и клетка. — 2005. — Т. 21, № 2. — С. 145-150. — Бібліогр.: 14 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860187989299167232 |
|---|---|
| author | Kyrylenko, T.K. Martynenko, O.I. Alkhimova, O.G. |
| author_facet | Kyrylenko, T.K. Martynenko, O.I. Alkhimova, O.G. |
| citation_txt | Nuclear genome size and karyotype analysis in Papaver for BAC library construction / T.K. Kyrylenko, O.I. Martynenko, O.G. Alkhimova // Биополимеры и клетка. — 2005. — Т. 21, № 2. — С. 145-150. — Бібліогр.: 14 назв. — англ. |
| collection | DSpace DC |
| container_title | Біополімери і клітина |
| description | The objective of the research carried out is study of the pathway of alkaloid production in Papaver species and cell lines, and integration data on physical mapping of newly developed marker DNA sequences with existing difference in expression of genes for key enzymes of alkaloid biosynthesis. This research requires the knowledge on genome structure and organization, and development of genomic resources for detailed characterization of opium poppy genome. The work is focused on the investigation of some features of genome organization of Papaver somniferum and related species, Papaver bracteatum and Papaver rhoeas. These characteristics are necessary for the construction of a BAC library which would be used as appropriate genomic resource for characterization of karyotype changes in poppy stocks with altered alkaloid biosynthesis pathway. Some of these stocks (cell lines) were generated and differed in the types of alkaloids they accumulated.
Роботу присвячено вивченню шляхів біосинтезу алкалоїдів у видів Papaver та інтегруванню даних фізичного картування нових маркерних послідовностей ДНК з відмінностями в експресії генів ключових ферментів біосинтезу алкалоїдів. Таке дослідження потребує знання структури геному та його організації, а також передбачає наявність геномних ресурсів задля детального аналізу геному опійного маку. У даному повідомленні визначено розмір геному опійного маку та зроблено каріотипічний аналіз Papaver somniferum і його споріднених видів, P. bracteatum і P. rhoeas. Отримані характеристики є необхідними для створення ВАС бібліотеки, яка слугує базисом для цитогенетичного картування та пошуку змін каріотипу в лініях маку, що відрізняються за шляхами біосинтезу алкалоїдів. Вже одержано декілька клітинних ліній, які накопичують різні типи алкалоїдів.
The objective of the research carried out is study of the pathway of alkaloid production in Papaver species and cell lines, and integration data on physical mapping of newly developed marker DNA sequences with existing difference in expression of genes for key enzymes of alkaloid biosynthesis. This research requires the knowledge on genome structure and organization, and development of genomic resources for detailed characterization of opium poppy genome. The work is focused on the investigation of some features of genome organization of Papaver somniferum and related species, Papaver bracteatum and Papaver rhoeas. These characteristics are necessary for the construction of a BAC library which would be used as appropriate genomic resource for characterization of karyotype changes in poppy stocks with altered alkaloid biosynthesis pathway. Some of these stocks (cell lines) were generated and differed in the types of alkaloids they accumulated.
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| first_indexed | 2025-12-07T18:05:11Z |
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ISSN 0233-7657. Біополімери і клітина. 2005. T. 21. № 2
Г Е Н О М І Й О Г О РЕГУЛЯЦІЯ
Nuclear genome size and karyotype analysis in
Papaver for ВАС library construction
Т. K. Kyrylenko, О. I. Martynenko, O. G. Alkhimova
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
150 Zabolotnoho str., 03143 Kyiv, Ukraine
E. mail: alkliimiv@hotmail.com
The objective of the research carried out is study of the pathway of alkaloid production in Papaver species
and cell lines, and integration data on physical mapping of newly developed marker DNA sequences with
existing difference in expression of genes for key enzymes of alkaloid biosynthesis. This research requires
the knowledge on genome structure and organization, and development of genomic resources for detailed
characterization of opium poppy genome. The work is focused on the investigation of some features of
genome organization of Papaver somniferum and related species, Papaver bracteatum and Papaver rhoeas.
These characteristics are necessary for the construction of a BAC library which would be used as
appropriate genomic resource for characterization of karyotype changes in poppy stocks with altered
alkaloid biosynthesis pathway. Some of these stocks (cell lines) were generated and differed in the types
of alkaloids they accumulated.
Key words: Papaver, alkaloids, genome size, bacterial artificial chromosome library, FISH.
Introduction. The data from several different alka
loid-producing plants suggest that their biosynthesis
and accumulation involve a highly regulated process
that includes cell-, tissue-, development and envi
ronment-specific controls [1, 2] . The evolution of
alkaloid pathways together with their cellular com-
partmentation appears to be closely associated with
the primary reactions from which they have evolved.
Opium poppy, Papaver somniferum, is cultivated for
its alkaloid-rich latex. Tyrosine decarboxylase
(TyDC) is the first enzyme in poppy alkaloid bio
synthesis and is encoded by a small gene family.
Members of this family are differentially expressed in
organs of the plant and cultivated cells [3 ]. With the
availability of an increasing number of genes involved
in alkaloid biosynthesis, increasing efforts would be
made to identify the regulators [4] associated with
the development of specialized cell types which relate
to alkaloid biosynthesis and accumulation. Gene loca-
© T. K. KYRYLENKO, O. I. MARTYNENKO, O. G. ALKHIMOVA,
2005
lization and isolation require a detailed knowledge of
genome structure. While genetic maps provide infor
mation on relative order of molecular markers and
genes along the chromosomes, physical mapping pro
vides data on the physical position of DNA sequence
within a genome. P. somniferum, P. bracteatum and
P. rhoeas belong to one taxonomic section within
genus Papaver and produce morphinan and bentho-
phenanthridine type alkaloids which belong to dif
ferent biosynthetic pathways.
Large-insert DNA libraries are one of the key
resources that facilitate gene isolation by positional
cloning and the analysis of genome organization,
structure and evolution. The easy handling and
propagation of the clones make BACs (bacterial
artificial chromosomes) an invaluable tool in genomic
research, used for a variety of applications, including
physical mapping and genome sequencing [5]. The
production of full-length cDNA molecules from geno
mic DNA libraries, representing genes of interest, is
of paramount importance in basic plant biology re-
145
mailto:alkliimiv@hotmail.com
K Y R Y L E N K O T . K. , M A R T Y N E N K O O . I . , A L K H I M O V A O . G
search as well as plant biotechnology [6]. Estimation
of genome size, karyotype parameters, testing restric
tion endonucleases patterns are the necessary charac
teristics for BAC library construction.
This study was undertaken to determine nuclear
genome size and genomic distribution of ribosomal
DNA loci and two families of repetitive DNA sequen
ces in Papaver species representing one taxonomic
section within genus Papaver, with the aim to expand
the number of species where these characteristics are
known, and aid in the construction of a BAC library
wich would be used as appropriate genomic resource
for characterization of karyotype changes in poppy
stocks with altered alkaloid biosynthesis pathway.
Material and Methods. Plant material. For DNA
extraction and chromosome slide preparations we
used seedlings and roots of poppy species, P. som-
niferum, P. bracteatum and P. rhoeas (accessions
from Kyiv Central Botanic Garden collection). DNA
was extracted from etiolated seedlings following Au-
subel et al. [7].
Determination of genome size. According to
Dolezel et al. [8 ], approximately 50 mg of midrib was
cut from a poppy, P. somniferum, young leaf and
transferred to a glass Petri dish. About 10 mg of a
young leaf of maize (Zea mays cv. CE-777) with 2C =
= 5.43 pg DNA was added and served as an internal
reference standard. The tissues were chopped simul
taneously in 1 ml of Otto I buffer (0.1 M citric acid,
0.5 % v/v Tween 20). Crude suspension of isolated
nuclei was filtered through a 50 pm nylon mesh.
Nuclei were then pelleted (300 g, 5 min), resus-
pended in 200 fil Otto I and incubated for 1 h at room
temperature. Finally, 600 fil Otto II buffer (0.4 M
Na 2 HP0 4 ) , supplemented with 50 [tg/ml RNAse and
50 i K g / m l propidium iodide (PI), was added. Samples
were analysed using Partec PAS flow cytometer
(«Partec GmbH», «Munster», Germany) equipped
with 488-nm argon laser. The gain of the instrument
was adjusted so that peak representing maize Gl
nuclei appeared approximately on channel 100 on
histogram of relative fluorescence intensity when
using 512-channel scale. About 5,000 nuclei were
analysed at rate 10—25 nuclei/s. Three plants were
measured per accession. Analysis of each plant was
repeated three times on different days. Nuclear DNA
content was calculated from individual measurements.
Fluorescence in situ hybridization (FISH). FISH
probe for ribosomal DNA was obtained by labelling a
pTa71 DNA clone containing 18S, 5.8S and 26S
rRNA genes [9] with biotin-16-dUTP («Roche»,
Germany) by Nick Translation. Along with rDNA
probe two new clones were used. These were made by
sonicating total genomic DNA from P. somniferum
(Danish flag accession) to an average length of 200 to
600 bp. The fragments were denatured at 100 °C in
0.1 M sodium phosphate buffer, pH 7.5, for 10 min
and incubated at 60 °C to C 0t of 0.02. The samples
were treated with SI nuclease. The resulting highly
repeated double-stranded DNA sequences were clo
ned into pUCJ8 at the PstI site. From several
thousand clones, 200 were chosen randomly for dot
hybridization with 3 2P-labelled total DNA from the
representatives of four Papaver taxonomic sections
(Meconella, Pilosa, MacranthalPapaver, Argemoni-
dium). Two clones, pPs21 and pPs41 gave very strong
signals with P. somniferum DNA and no visible
signals with three other sections members. These
clones were selected for further studies on in situ and
southern hybridizations. Digoxigenin-labelled probes
for these repeats were prepared using PCR with M13
direct and reverse primers and pPs21 and pPs41
clones as a templates.
Metaphase spreads were prepared according to
Alkhimova et al. [10]. The slides were treated with
100 mg/ml RNAse in a 2 x SSC solution at 37 °C for
1 h in a humid chamber, washed 3 x 5 min in 2 x SSC
at room temperature. After two washes in 2 x SSC the
slides were treated in 4 % paraformaldehyde for
10 min at room temperature, washed in 2 x SSC,
dehydrated in ethanol series, and air dried. Prior to
hybridization, the probes were mixed in a solution
containing 50 % formamide, 10 % dextran sulphate,
0.12 % SDS in 2 x SSC and 50 ng/,ul salmon sperm
DNA. 1 fil of probe in 30 fil hybridization mixture per
slide was used. The hybridization mixture was dena
tured at 70 °C for 10 min and incubated on ice for
10—15 min before being added to the preparations.
The chromosomes together with the probes were
denatured at 70 °C for 5 min and the hybridization
was performed overnight at 37 °C in a humid cham
ber. The slides were then washed in 2 x SSC at 42 °C
and rinsed in a stringent washing solution of 20 %
formamide in 0.1 x SSC at 42 °C for 10 min, followed
by several washes in 2 x SSC and 4 x SSC (0.2 %
Tween). The sites of digoxigenin- and biotin-labelled
probe hybridization were detected using anti-digo-
xigenin fluorescein («Roche») and streptavidin conju
gated to Cy3 («Sigma», USA), respectively. Finally,
the preparations were counterstained with DAPI (0.2
146
N U C L E A R G E N O M E S I Z E A N D K A R Y O T Y P E ANALYSIS IN P A P A V E R
120 -I
100 -
| 8 0 '
If 6 0 '
-o
I 40-
20 -
0 -
0
( W G / m l ) and mounted in Vectashield antifade solution
(«Vector Laboratories*, USA).
The preparations were evaluated using Olympus
BX60 microscope equipped with optical filter sets
appropriate for DAPI, fluorescein and Cy3 fluo
rescence. The images of DAPI, fluorescein and Cy3
fluorescence were acquired separately with a b / w
CCD camera, which was interfaced to a PC running
the ISIS software («Metasystems», «Altlussheim»,
Germany) . The images were superimposed after con
trast and background optimization.
Results and Discussion. Estimation of genome
size of Papaver somniferum. Flow cytometry is a rapid
technique that allows accurate estimation of nuclear
DNA content [11] . To determine nuclear DNA con
tent in absolute units , fluorescence intensity of nuclei
is compared with that of nuclei isolated from a species
with known nuclear genome size. Fluorescence of
Pl-stained nuclei were analysed using a Partec PAS
II flow cytometer. Nuclei isolated from Z. mays cv.
CE-777 with known nuclear genome size (2C =
= 5.43 pg DNA) were used as internal s tandard to
estimate nuclear DNA content of P. somniferum in
absolute units. Small amounts of leaf tissues (stan
dard and sample) were simultaneously chopped in
buffer, supplemented with PI and RNAse. Suspension
of nuclei was filtered through 50 p.m nylon and stored
on ice prior to analysis . 2C DNA content of P.
somniferum was calculated according to formula:
2C nuclear DNA content =
5.43 * G, peak mean of Papaver r t r
= ~ — : r^r = 6.46 pg.
G, peak mean of Zea
Genome size, which represent one copy of nuc
lear DNA equal to 1C, was further determined as:
Genome size =
= 2C nuclear DNA content (pg) . Q 9 7 8 . 1 Q 9 B P R L L ]
Fig. 1 . Estimation of nuclear DNA
content using flow cytometry. Si
multaneous analysis of nuclei iso
lated from Zea mays U) (2n -
- 22) used as internal standard
and from Papaver somniferum
(2)
The results of the study showed (Fig. 1) that the
size of P. somniferum nuclear genome equal 6.46 pg
is smaller than previously estimated [12].
Karyotype of P. somniferum and related species,
P. bracteatum and P. rhoeas. A karyotype, which is
characterized by the number and morphology of
chromosomes, is an important characteristic of a
species. Methods for chromosome preparation and in
situ hybridization essentially followed Heslop-Harri-
son et al. [13] . Root tips were fixed, partially
digested with enzymes and cells were spread on
slides. At least ten well-spread metaphase plates with
similar degree of chromatin condensation were used
to make chromosomal measurements . For constructing
the karyotype, the chromosomes were arranged in
order of decreasing size and increasing asymmetry
(Fig. 2) .
Localization of rDNA loci and repetitive DNA
clones. Fluorescence in situ hybridization on P. som
niferum chromosomes showed that pPs21 and pPs41
DNA sequences were distributed mostly uniformly
along all chromosomes with gaps near centromeres
and nucleolar organizer regions (data not shown).
FISH with P. bracteatum, the representative of the
same taxonomic group Macranthal Papaver, is shown
in Fig. 3. DNA sequences have been shown to be
dispersed over the P. bracteatum genome. It had
substantially more copies of pPs41 dispersed along
most chromosomes (Fig. 3, b) than pPs21 which gave
less number of dots.
Preparation of BAC library construction. BAC
library is a source of DNA clones, which will be used
as landmarks for chromosome identification, and also
for the search of clones consisted of single copy
sequences. An important step toward the structural
analysis of a functional DNA domains is the const
ruction of a large-insert libraries. Their inserts repre
sent large DNA fragments that can be easily localized
on mitotic chromosomes using FISH and allow selec-
147
5 yon
c
Fig. 2. Karyoidiogram of Papaver somniferum (a), P. bracteatum
(b) and P. rhoeas (c) showing the length of individual chromosomes
and centromere position. Distribution of r D N A loci revealed by in
situ hybridization with pTa71 probe
t ion of BAC clones with def ic iency in repetitive D N A
sequences . T h i s approach requires determinat ion of
restriction e n d o n u c l e a s e s su i table for library const
ruct ion, vector se lec t ion , appropriate ratio of high
molecular weight D N A a n d c h o s e n vector, optimal
l igation and transformation condi t ions .
Testing conditions for different restriction en
zymes. EcoRI, BamHI, or Hindlll, the restriction
e n z y m e s most frequently u s e d in BAC cloning [5].
H i g h molecular weight D N A of P. somniferum, obtai
n e d by purification of protoplasts from the seed l ings
in e n z y m e solut ion fo l lowed by e m b e d d i n g on low-
melt ing-point agarose , was d iges ted with the five
restriction e n z y m e s , which can be ranged according to
Fig. 3. In situ hybridization showing the localization of repetitive
sequences, pPs21 (a) and pPs41 (b), derived from opium poppy, on
chromosomes of Papaver bracteatum. Arrows mark NORs , arrow
heads indicate major pPs21 sites at the terminal region of two
chromosomes
increase of recognit ion s i tes , in the nex t order: Kpnl,
Xbal, BamHI, EcoRI, Hindlll. As Hindi 11 restriction
fragments conta ined T y D C sequence (hybridizat ion
data not s h o w n ) , and Hindlll d igest ion gave more
fragments , this e n z y m e is c h o s e n for the construction
of P. somniferum BAC library.
Logistics. T h e p lnd igo B A C - 5 (Hind11Y-Cloning
R e a d y ) vector has been chosen for the P. somniferum
BAC library construct ion. T h e vector is provided in a
«ready- to -use» state . It has been l inearized at the
unique restriction e n z y m e recognit ion site (Hindlll),
dephosphory la ted and rigorously tes ted for purity and
recombinant c loning eff iciency (Epicentre) . C o m p e
tent Escherichia coli D H 1 0 B cel ls («Gibco BRL»,
USA) and Gibco BRL Cel l -Porator Sys tem («Life
Techno log ies* , U S A ) have b e e n u s e d to get effective
and rel iable result. T h e vector to D N A ratio has been
calculated to be in proportion approximate ly 1:10.
Conclusions drawn. T h e cultured cell l ines which
accumulated alkaloids of only o n e type , morphinan
( thebaine) or benthophenanthr id ine (sanguinarine)
have been generated from P. bracteatum (Fig. 4 ) . As
the members of the T y D C g e n e family are dif feren
tially expres sed in cel ls and organs accumulat ing
predominant alkaloid type [ 1 ] , o n e can a s s u m e that
different g e n e s of this family are «responsible» for the
alkaloid type produced. Southern hybridizat ion with
T y D C probe have revealed the different organization
of restriction fragments in unorganized callus culture
[3, 1 4 ] . T h e opium poppy T y D C g e n e family is
composed of about 15 m e m b e r s that are divided into
two subfamil ies with regard to the sequence identity
[3 ]. T h e s e two groups d is t inguish in organ-specif ic
express ion , namely , the m e m b e r s of the T y D C l - l i k e
subgroup are e x p r e s s e d abundant ly in roots , whi le the
members of the T y D C 2 - l i k e subgroup — in roots and
1 4 8
Fig. 4. Thin-layer chromatographic analysis of alkaloids in Papaver
bracteatum cultured cell clones: 1—4 — primary cell cultures (up to
5 months in culture); EM, EM5, EMIO — morphogenous lines; CA
— dedifferentiated callus. Authentic standards of sanguinarine (S),
protopine (P), allocryptopine (A) and thebaine (F) were also run
stems. The members of the P. somniferum TyDC
gene family show also differences in the develop
mental, tissue-specific, and inducible expression pat
terns. This gene family can be used as a molecular
marker to study the regulation and localization of
alkaloid biosynthesis in the poppy species.
Fluorescence in situ hybridization has been per
formed to investigate the physical distribution of
repetitive clones along the chromosomes of P. somni
ferum and P. bracteatum species. Two new clones
have been used, pPs21 and pPs41. In situ hybridi
zation on P. somniferum chromosomes has shown that
pPs21 and pPs41 DNA sequences are distributed
mostly uniformly along all chromosomes with gaps
near centromeres and nucleolar organizer regions
(data not shown). These repetitive sequences have
been shown to be dispersed over the P. bracteatum
genome distinguishing by the copy numbers of their
repeats (Fig. 3). For the first time, the localization of
the ribosomal DNA on P. bracteatum chromosomes is
shown.
BAC library is a source of new DNA clones,
which will be used as landmarks for the chromosome
identification, and also for searching the clones con
sisted of single copy sequences. An important step
toward the structural analysis of functional DNA
domains is the construction of large-insert libraries.
N U C L E A R G E N O M E S I Z E A N D K A R Y O T Y P E ANALYSIS IN P A P A V E R
The construction of the representative ВАС library
requires the determination of the genome size to
obtain the appropriate amount of clones. The results
of the study show that the size of the P. somniferum
nuclear genome equal to 6.46 pg is smaller than
previously estimated. The genome size of P. somni
ferum (In = 22) which represents one copy of nuclear
DNA equal to 1C, has been determined as 3100 Mb.
Based on this haploid genome size and average insert
size of fragments around 150 kb that can be cloned
efficiently in the ВАС vector [5], the library repre
senting five genome equivalents would consist of
100000 clones. Using cDNA probes will allow the
verification of the coverage. The results obtained will
be used for the construction of the large insert size
genomic library as a new molecular resource.
Acknowledgements. We are grateful to Dr J.
Dolezel (Institute of Experimental Botany, Olomouc,
Czech Republic) for providing us with technical faci
lities, and P. Suchankova for technical assistance.
This work was supported by the National Aca
demy of Sciences of Ukraine and the International
Atomic Energy Agency (Research Contract No.
12235/RBF).
T. K. Кириленко, О. 1. Мартиненко, О. Г. Алхімова
Визначення розміру ядерного геному і каріотипічний аналіз
видів Papaver для створення ВАС бібліотеки
Резюме
Роботу присвячено вивченню шляхів біосинтезу алкалоїдів у
видів Papaver та інтегруванню даних фізичного картування
нових маркерних послідовностей ДНК з відмінностями в
експресії генів ключових ферментів біосинтезу алкалоїдів.
Таке дослідження потребує знання структури геному та його
організації, а також передбачає наявність геномних ресурсів
задля детального аналізу геному опійного маку. У даному
повідомленні визначено розмір геному опійного маку та зроб
лено каріотипічний аналіз Papaver somniferum і його спорід
нених видів, P. bracteatum і P. rhoeas. Отримані характеристи
ки є необхідними для створення ВАС бібліотеки, яка слугує
базисом для цитогенетичного картування та пошуку змін
каріотипу в лініях маку, що відрізняються за шляхами біоси
нтезу алкалоїдів. Вже одержано декілька клітинних ліній, які
накопичують різні типи алкалоїдів.
Ключові слова: Papaver, алкалоїди, розмір геному, ВАС
бібліотека, флуоресцентна гібридизація in situ.
Т. К Кириленко, Е. И. Мартиненко, Е. Г. Алхимова
Определение размера ядерного генома и кариотипический
анализ видов Papaver для создания ВАС библиотеки
Резюме
Робота посвящена изучению пути биосинтеза алкалоидов у
видов Papaver и интегрированию данньїх по физическому кар-
1 4 9
KYRYLENKO Т. К., MARTYNENKO О. I., ALKHIMOVA О. G.
тированию новых маркерных последовательностей ДНК с
различиями в экспрессии генов ключевых ферментов биосинте
за алкалоидов. Такое исследование нуждается в знании струк
туры генома и его организации, а также предполагает наличие
геномных ресурсов для детального анализа генома опийного
мака. В данном сообщении определен размер генома опийного
мака и сделан кариотипический анализ Papaver somniferum и
его родственных видов, P. bracteatum и P. rhoeas. Полученные
характеристики необходимы для создания ВАС библиотеки,
предоставляющей базис для цитогенетического картирования
и поиска изменений кариотипа у линий мака, различающихся
путями биосинтеза алкалоидов. Уже выделены несколько кле
точных линий, накапливающих разные типы алкалоидов.
Ключевые слова: Papaver, алкалоиды, размер генома, ВАС
библиотека, флуоресцентная гибридизация in situ.
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УДК 575:577.21.12
Надійшла до редакції 18.12.03
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| id | nasplib_isofts_kiev_ua-123456789-155232 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| language | English |
| last_indexed | 2025-12-07T18:05:11Z |
| publishDate | 2005 |
| record_format | dspace |
| spelling | Kyrylenko, T.K. Martynenko, O.I. Alkhimova, O.G. 2019-06-16T12:23:27Z 2019-06-16T12:23:27Z 2005 Nuclear genome size and karyotype analysis in Papaver for BAC library construction / T.K. Kyrylenko, O.I. Martynenko, O.G. Alkhimova // Биополимеры и клетка. — 2005. — Т. 21, № 2. — С. 145-150. — Бібліогр.: 14 назв. — англ. DOI: http://dx.doi.org/10.7124/bc.0006E5 https://nasplib.isofts.kiev.ua/handle/123456789/155232 575:577.21.12 The objective of the research carried out is study of the pathway of alkaloid production in Papaver species and cell lines, and integration data on physical mapping of newly developed marker DNA sequences with existing difference in expression of genes for key enzymes of alkaloid biosynthesis. This research requires the knowledge on genome structure and organization, and development of genomic resources for detailed characterization of opium poppy genome. The work is focused on the investigation of some features of genome organization of Papaver somniferum and related species, Papaver bracteatum and Papaver rhoeas. These characteristics are necessary for the construction of a BAC library which would be used as appropriate genomic resource for characterization of karyotype changes in poppy stocks with altered alkaloid biosynthesis pathway. Some of these stocks (cell lines) were generated and differed in the types of alkaloids they accumulated. Роботу присвячено вивченню шляхів біосинтезу алкалоїдів у видів Papaver та інтегруванню даних фізичного картування нових маркерних послідовностей ДНК з відмінностями в експресії генів ключових ферментів біосинтезу алкалоїдів. Таке дослідження потребує знання структури геному та його організації, а також передбачає наявність геномних ресурсів задля детального аналізу геному опійного маку. У даному повідомленні визначено розмір геному опійного маку та зроблено каріотипічний аналіз Papaver somniferum і його споріднених видів, P. bracteatum і P. rhoeas. Отримані характеристики є необхідними для створення ВАС бібліотеки, яка слугує базисом для цитогенетичного картування та пошуку змін каріотипу в лініях маку, що відрізняються за шляхами біосинтезу алкалоїдів. Вже одержано декілька клітинних ліній, які накопичують різні типи алкалоїдів. The objective of the research carried out is study of the pathway of alkaloid production in Papaver species and cell lines, and integration data on physical mapping of newly developed marker DNA sequences with existing difference in expression of genes for key enzymes of alkaloid biosynthesis. This research requires the knowledge on genome structure and organization, and development of genomic resources for detailed characterization of opium poppy genome. The work is focused on the investigation of some features of genome organization of Papaver somniferum and related species, Papaver bracteatum and Papaver rhoeas. These characteristics are necessary for the construction of a BAC library which would be used as appropriate genomic resource for characterization of karyotype changes in poppy stocks with altered alkaloid biosynthesis pathway. Some of these stocks (cell lines) were generated and differed in the types of alkaloids they accumulated. Acknowledgements. We are grateful to Dr J. Dolezel (Institute of Experimental Botany, Olomouc, Czech Republic) for providing us with technical facilities, and P. Suchankova for technical assistance. This work was supported by the National Academy of Sciences of Ukraine and the International Atomic Energy Agency (Research Contract No.
 12235/RBF). en Біополімери і клітина Геном та його регуляція Nuclear genome size and karyotype analysis in Papaver for BAC library construction Визначення розміру ядерного геному і каріотипічний аналіз видів Papaver для створення ВАС бібліотек Определение размера ядерного генома и кариотипический анализ видов Papaver для создания ВАС библиотеки published earlier |
| spellingShingle | Nuclear genome size and karyotype analysis in Papaver for BAC library construction Kyrylenko, T.K. Martynenko, O.I. Alkhimova, O.G. Геном та його регуляція |
| title | Nuclear genome size and karyotype analysis in Papaver for BAC library construction |
| title_alt | Визначення розміру ядерного геному і каріотипічний аналіз видів Papaver для створення ВАС бібліотек Определение размера ядерного генома и кариотипический анализ видов Papaver для создания ВАС библиотеки |
| title_full | Nuclear genome size and karyotype analysis in Papaver for BAC library construction |
| title_fullStr | Nuclear genome size and karyotype analysis in Papaver for BAC library construction |
| title_full_unstemmed | Nuclear genome size and karyotype analysis in Papaver for BAC library construction |
| title_short | Nuclear genome size and karyotype analysis in Papaver for BAC library construction |
| title_sort | nuclear genome size and karyotype analysis in papaver for bac library construction |
| topic | Геном та його регуляція |
| topic_facet | Геном та його регуляція |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/155232 |
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