MITOTIC TRANSMISSION OF RIBOSOMAL CHROMATIN IN EMBRYONIC PIG KIDNEY CELLS (PK CELL LINE)
V. N. Stefanova,1, 2 S. Yu. Demin 2
1 All-Russian Institute of Genetics and Farm Animal Breeding, RAA, St. Petersburg-Pushkin, and
2 Institute of Cytology, RAS, St. Petersburg;
1 e-mail: vestefan@mail.ru
Mammalian nucleolar organizers (NORs) contain ribosomal (rRNA) genes associated with argyrophilic proteins and
can be specifically stained by silver. These genes are clustered in four loci of PK meta- and submetacentric
chromosomes 8 and 10. According to our data reported elsewhere, one of PK NORs contains a large amplification of
rRNA that is manifested on silver stained chromosomes as abnormally large Ag+-NOR 8a. It is proposed
that such a redundancy of rRNA genes induced their dose compensation in the form of transcriptional silencing,
triggering the rise of Ag-negative ribosomal chromatin. To test this assumption, we carried out a morphometrical
analysis of Ag+-NOR (bearing) chromosomes from prometaphase sets with modal chromosome number. We found
that in the individual chromosome set a longer homologue 10L of chromosome 10 had always a larger
Ag+-NOR area than did a shorter homologue 10S. Thus, PK karyotype consists of two pairs of
heteromorphous Ag+-NORs: 8a and 8, 10L and 10S. One half of tested chromosome sets
revealed four Ag+-NOR chromosomes, the other half had one Ag-negative NOR nearly equally belonging to
chromosome either 8 or 10S. The majority of prometaphase Ag+-NORs showed partial or full
chromatid splitting that allows for Ag+-NOR area measurements of (sister) chromatid. The area ratio of
larger to smaller chromatid Ag+-NOR strongly varied for all chromosomes except chromosomes 8a. The
maximum value of this ratio reached 5.1 for chromosome 8 and 3.4, and 2.3 - for 10L and
10S (vs. 1.6 for 8a). Proportions of chromosomes with the ratio 1.25 and more, were: near two thirds -
among chromosomes 8, near half - among 10L and 10S, but less than a quarter - among
chromosomes 8a. These findings show that progressive differentiation of sister chromatid NORs in regard of the
content of Ag-negative ribosomal chromatin may lead to an unequal Ag+-NOR distribution between daughter
cells. To test this prediction, we developed a new technique for obtaining two dimensional (2D) preparations of
stretched PK cells, which makes it possible to avoid the stage of hypotonic treatment of living cells, since this
treatment levels the silver staining of NORs and prenucleolar bodies of fixed telophase cells. We found that some
daughter nuclei from early telophase cells revealed the value of Ag+-NOR separation equal to 4 : 3
instead of the common value equal to 4 : 4. Complimentary 2D FISH with 28S + 18S mink rDNA probe showed that early
telophase rRNA loci, detected as four bright large spots, are close by area and shape to Ag+-NORs of the
corresponding cells. Sometimes, however, one of the daughter nuclei showed three such domains, in addition to one
slight linear FISH signal that most probably represented Ag-negative NOR. A delayed separation of sister chromatids
is the main structural characteristic of inactive chromatin (Azuara et al., 2003). It was established that the
largest PK Ag+-NOR (chromosome 8a) showed a high level of cohesion and (or) twisting of sister
chromatids that is characteristic of prophase rather than prometaphase PK chromosomes. These findings, together
with the above cited literature data, give evidence for enrichment of Ag+-NOR by either inactive or
(and) low active late-replicating chromatin.
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