Документ взят из кэша поисковой машины. Адрес оригинального документа : http://zmmu.msu.ru/rjt/articles/ther6_1%20007_013%20Zhdanova.pdf Дата изменения: Sat Feb 2 18:23:12 2008 Дата индексирования: Tue Oct 2 02:04:09 2012 Кодировка: Windows-1251

Russian J. Theriol. 6 (1): 713

ї RUSSIAN JOURNAL OF THERIOLOGY, 2007

The distributions of telomeric and ribosomal DNA on the chromosomes of two closely related species, Sorex araneus and Sorex granarius (Soricidae, Eulipotyphla)
Natalia S. Zhdanova*, Julia M. Minina, Tatiana V. Karamysheva & Nikolai B. Rubtsov
ABSTRACT. It is widely believed that Robertsonian rearrangements have played a key role in the chromosome evolution of species of the Sorex araneus group. We present FISH data relating to the distribution of telomeric repeats and 18S rDNA on the chromosomes of Sorex araneus and Sorex granarius, which have karyotypes with almost identical chromosome arms. All chromosomes in S. araneus (Novosibirsk race) are metacentrics, whereas S. granarius has an acrocentric karyotype with two metacentric exceptions. In FISH experiments we revealed telomeric repeats at the ends of all S. araneus chromosomes but only on the short arms of S. granarius acrocentrics, which, as we have shown earlier, amount up to 300 kb in length. FISH signals of the (TTAGGG)n probe and the probe derived by microdissection of the pericentric regions of S. granarius acrocentrics a and b were co-localised or sequentially localised on distinct chromatin fibres of S. granarius. 18S rDNA clusters were found at the ends of short arms of 12 out of 16 S. granarius acrocentric pairs. In S. araneus primary cell culture fibroblasts rDNA was found at the ends of the q, t and u arms. However, after long cultivation of these cells an additional FISH signal of rDNA was found at the distal end of the o arm of chromosome go. In some regions the FISH signal of rDNA coincided with the signal of the telomeric probe. We suppose that rapid concerted evolution of telomeric and rDNA led to the repatterning of these repetitive DNA fractions in the sibling species S. araneus and S. granarius as well as the formation of large telomeres with unusual structure at the ends of the S. granarius chromosomes. KEY WORDS: Shrew chromosomes, FISH, large telomeres, ribosomal DNA.
Natalia S. Zhdanova [zhdanova@bionet.nsc.ru], Julia M. Minina [minina jul@mail.ru], Tatiana V. Karamysheva [kary@bionet.nsc.ru], and Nikolai B. Rubtsov [rubt@bionet.nsc.ru], Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.

Распределение теломерной и рибосомальной ДНК на хромосомах двух близкородственных видов, Sorex araneus и Sorex granarius (Soricidae, Eulipotyphla)
Н.С. Жданова, Ю.М. Минина, Т.В. Карамышева, Н.Б. Рубцов
РЕЗЮМЕ. Полагают, что Робертсоновские перестройки играли важную роль в эволюции кариотипов видов группы S. araneus. В этой связи проанализированы данные о распределении теломерных повторов и 18S рибосомальной ДНК в хромосомах двух видов-двойников, Sorex araneus и Sorex granarius, чьи кариотипы составлены из практически идентичных плеч. Все хромосомы Sorex araneus (Новосибирская раса) являются метацентриками, тогда как кариотип Sorex granarius является полностью акроцентрическим, за исключением двух пар метацентриков. Теломерные повторы были выявлены на концах всех хромосом S. araneus, а в хромосомах S. granarius только на концах коротких плеч акроцентриков, где они достигают размера 300 т.п.н. Используя пробу к теломерным повторам и микродиссекционную пробу к перицентромерным районам хромосом a и b S. granarius, мы выявили ко-локализацию и последовательную локализацию сигналов этих ДНК проб на хроматиновых фибриллах S. granarius. Кластеры 18S рДНК были обнаружены в коротких плечах 12 из 16 пар акроцентриков S. granarius и на концах длинных плеч q, t и u у S. araneus. Однако при длительном культивировании фибробластов одной из особей S. araneus дополнительные кластеры 18S рДНК были выявлены в дистальной части плеча o хромосомы go. На некоторых хромосомах сигналы FISH теломерной и рДНК совпадали. По-видимому, быстрая амплификация и перераспределение теломерной и рДНК в хромосомах S. araneus и S. granarius, а также формирование 'длинных' теломер с необычной структурой в коротких плечах акроцентриков S. granarius является следствием совместной эволюции этих типов повторов в ходе недавней реорганизации кариотипов землероек. КЛЮЧЕВЫЕ СЛОВА: хромосомы землеройки, FISH, длинные теломеры, рибосомная ДНК.
* Corresponding author


&

N.S. Zhdanova, J.M. Minina, T.V. Karamysheva & N.B. Rubtsov

Introduction
Numerous data show that repetitive sequences including telomeric and ribosomal DNA can be involved in the evolution of mammalian chromosomes. The size of telomeric and rDNA clusters is, to a large extent, genetically determined (Zhu et al., 1998; Manning et al., 2002). (TTAGGG)n repeats are located mainly at the chromosome termini in so called telomeres and preserve the integrity of the cell genome, while telomere shortening or erosion leads to chromosomal instability and rearrangements. As well as telomeres, (TTAGGG)n have been found at interstitial chromosome sites (ITs) (Meyne et al., 1990). ITs can be the remnants of ancestral chromosome fusions (Azzalin et al., 2001; Ruiz-Herrera et al., 2002; Hartmann & Scherthan, 2004) and can increase chromosomal instability where they occur (Mondello et al., 2000; Kilburn et al., 2001; Rivero et al., 2004). The presence of telomeric repeats can probably lead to chromosome rearrangements in both directions: fusions and fissions. rDNA is also often involved in chromosome reorganization. Closely related species and even chromosome races of the same species differ in number and location of rDNA clusters (Dobigny et al., 2003). Nevertheless, only scanty information is available on the role and mechanisms underlying the redistribution of telomeric and rDNA during chromosomal evolution. The fusion of acrocentrics (Robertsonian fusion) and the reverse process (Robertsonian fission) are among the most common rearrangements which took place during karyotypic evolution in mammals. Thus in the Sorex araneus group they played an especially important role (Wуjcik & Searle, 1988; Searle, 1993). The study of repetitive DNA in the vicinity of evolutionary breakpoints may elucidate the mechanisms of Robertsonian (Rb) rearrangements. Here we present data on the distribution of telomeric repeats and rDNA on chromosomes of two closely related species: Sorex araneus (Novosibirsk race) and Sorex granarius that diverged a few hundred thousands years ago (Taberlet et al., 1994). Their karyotypes have almost identical chromosome arms, differing only in the number of metacentrics (Wуjcik & Searle, 1988). The comparative study of S araneus and S. granarius chromosomes provides an opportunity to clarify the role of repetitive DNA in karyotype evolution of mammals.

This paper summarises and extends upon the findings of a previously published work by Zhdanova et al. (2005).

Material and methods
The S. araneus and S. granarius primary fibroblast cell cultures were obtained from pieces of intercostal muscles. Metaphase chromosomes and distinct chromatin fibres for FISH and chromosome microdissection were prepared by standard techniques. A microdissected DNA probe was generated by DOP-PCR with the MW6 primer, and subsequent DNA labelling with biotin-16-dUTP or digoxigenin-11-dUTP was performed over additional PCR cycles (Rubtsov et al., 1996). The DNA probe used for detection of 18S rDNA (rDNA probe) was a 3.2-kb fragment of human 18S rDNA in pHr13 (Malygin et al., 1992). It was labelled with biotin-16-dUTP by nick translation. To visualize clusters of telomeric repeats the (TTAGGG)n probe labelled with biotin-11-dUTP (Ijdo et al., 1991) was used. FISH was performed according to a standard protocol with salmon sperm DNA as a carrier DNA. Biotin- and digoxigenin labelled probes were visualized with avidin-FITC and mouse antidigoxigenin antibodies conjugated to Cy3, respectively. Two-colour FISH with telomeric and microdissected DNA probes on distinct chromatin fibres was performed with suppression of dispersed repeats with shrew Cot1 DNA. Metaphase chromosomes and distinct fibres were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and analyzed using an Axioskop 2 Plus microscope (Zeiss, Germany) equipped with a CCD camera (CV M300, JAI Corporation, Japan), CHROMA filter sets, and the ISIS4 image-processing package of Metasystems GmbH in the Microscopic Centre of the Institute of Cytology and Genetics SB RAS, Novosibirsk. According to the nomenclature for S. araneus and S. granarius, the karyotypes in these species consist of chromosome arms named a u in decreasing order of size (Searle et al., 1991). Thus, in biarmed chromosomes the first letter denotes the long arm and the second corresponds to the short arm. Arm e is the homologue of the original mammalian X chromosome. The females are XX (de, de) and the males are XY1Y2. The Y1 is the true Y chromosome, whereas the Y2 is autosomal arm d (Pack et al., 1993). The Novosibirsk race of S. araneus shows metacentrics: af, bc, go, hn, ik, jl, mp, qr and tu (Wуjcik et al., 2003). In S. granar-

Figure 1. FISH on S. araneus (A) and S. granarius (B) chromosomes using a biotinylated telomeric probe generated by PCR. Chromosomes counterstained with DAPI. The S. araneus chromosomes on which ITs were detected are indicated. Metacentric chromosomes of S. granarius on which telomeric signals were not detected are indicated by arrows. FISH on S. granarius (C) and S. araneus (D) chromosomes, using a microdissected probe derived from the pericentric regions of six copies of the S. granarius chromosomes a and b. Chromosomes counterstained with DAPI. The S. araneus chromosomes on which signals were detected and metacentric chromosomes of S. granarius on which no signals were detected are indicated. Two-colour FISH (E) on S. granarius distinct chromatin fibres. The same fibres painted by biotinylated telomeric probe generated by PCR (1) and digoxigenin-11-dUTP labelled microdissected probe generated from the pericentric regions of six copies of S. granarius chromosomes a and b (2).


Telomeric and rDNA on shrew chromosomes

'




N.S. Zhdanova, J.M. Minina, T.V. Karamysheva & N.B. Rubtsov In S. granarius FISH with a telomeric probe showed strong signals only on the short arms of acrocentrics (Fig. 1B). Earlier we showed that the short arms of acrocentrics contain telomeric repeats up to 300 kb in length (213 kb on average), while the other telomeres were on average 3.8 kb in length (Zhdanova et al., 2005). To our knowledge, telomeres about 300 kb in size have not hitherto been described in mammals. The longest telomeres previously detected (up to 150 kb) were found in some strains of laboratory mice. However, the size of telomeres in wild mice (M. musculus and M. spretus) does not exceed 25 kb and 515 kb, respectively. It is believed that inbreeding can result in the elongation of mouse telomeres by means of an unknown mechanism (Zijlmans et al., 1997; Zhu et al., 1998, Manning et al., 2002). Small differences in telomere lengths between the long and short arms of mammalian chromosomes have been described previously, and longer telomeres are usually located on the long arms of chromosomes (Zijlmans et al., 1997; Slijepcevic, 1998). Trying to explain this phenomenon, Slijepcevic (1998: 136140) suggested that the difference in telomere size is caused by competitive telomere/centromere relations at the bouquet stage of meiosis. At this stage high tension and breakages in regions between telomeres and centromeres can be generated when long telomeres are located in short arms. The telomere organization in S. granarius does not agree with this hypothesis.

ius, only two pairs are metacentrics: de and tu. Other chromosomes are acrocentrics. The GTG-banding patterns of the chromosome arms of the two species are nearly identical (Wуjcik & Searle, 1988; Volobouev & Dutrillaux, 1991).

Results and discussion Distribution of telomeric repeats on chromosomes of S. araneus and S. granarius
FISH with the telomeric DNA probe gave distinct signals at all telomeres of the chromosomes of S. araneus. Signals corresponding to ITs were observed mostly at the pericentric regions of metacentrics go, hn, ik, jl, mp and qr. The frequencies of FISH signals at ITs varied from 29% for chromosome jl to 81% for chromosome mp. However, in pericentric regions of metacentrics af, bc, de and tu, the frequencies of signals did not exceed 10%. In addition, signals were also detected on arms a and b of chromosomes af and bc with frequencies of 19% and 12%, respectively (Fig. 1A). Sorex araneus displays a surprising degree of chromosomal variation due to Robertsonian fusions. Only metacentrics af, bc, de and tu were present in the common ancestor of all races of S. araneus. These metacentrics were formed before race formation (Wуjcik et al., 2003). The FISH experiments showed that the number of TTAGGG repeat copies at pericentric regions of these old metacentrics is lower than those of other biarmed chromosomes go, hn, ik, jl, mp and qr (see also Zhdanova et al., 2005). The latter are younger. They originated from acrocentrics during chromosomal race formation. Metacentric jl is younger than af, bc, de, and tu, but older than go, hn, ik, mp and qr, since it is found in all races of S. araneus but absent in other species of the S. araneus group. The chromosome jl has more copies of telomeric repeats at the pericentric region than old metacentrics but less than chromosomes go, hn, ik, mp and qr. The data obtained are most easily explained by the retention of (TTAGGG)n repeats after Robertsonian fusion in S. araneus, and by the loss or modification of telomeric repeats with time. It has been shown that in some mammalian species telomeric DNA is directly involved in Rb translocations (Metcalfe et al., 1998; Slijepcevic, 1998; Castiglia et al., 2002). Contrary to that, in the house mouse breakpoints associated with Rb translocation are localized within subtelomeric regions containing sat-DNA (Garagna et al., 2002). Probably Rb fusions can be the result of nonhomologous crossing over within telomeric or subtelomeric chromosomal regions enriched with repetitive DNA.

FISH on metaphase chromosomes and distinct chromatin fibres using telomeric and microdissected DNA probes
A microdissected probe was generated from 6 copies of the pericentric regions of S. granarius chromosomes a and b. In S. granarius it painted the regions which were also detected by the telomeric DNA probe (Fig. 1C) whereas in S. araneus chromosomes it only painted the pericentric regions of arms a and b of af and bc (Fig. 1D). Two-colour FISH on distinct chromatin fibres of S. granarius revealed regions painted either with telomeric and microdissected probes or only with one of them (Fig. 1E).

Distribution of 18S rDNA sequences on S. araneus and S. granarius chromosomes
In S. araneus fibroblasts from primary cell cultures, 18S rDNA was revealed at the ends of the q, t and u arms of chromosomes qr and tu (Fig. 2A). However, additional clusters of rDNA at the ends of the o arms

Figure 2. Two-colour FISH on S. araneus (A, B) and S. granarius (C, D) chromosomes, using biotinylated probe of 18S rDNA (A, D) and digoxigenin-11-dUTP labelled telomeric probe (B, C). Typical profiles of relative signal intensities along acrocentric m chromosome of S. granarius (E) and qr chromosomes S. araneus (F) from the proximal telomere (left) to the distal telomere (right). The maximum signal in the analysed image is given as 100%. 1 DAPI staining of chromosome; 2 profile of 18S rDNA probe; 3 profile of telomeric probe.


Telomeric and rDNA on shrew chromosomes






N.S. Zhdanova, J.M. Minina, T.V. Karamysheva & N.B. Rubtsov

were revealed after long (about one year) cultivation of these cells. Previously, Ag-positive material was found at the ends of the q, t, o and u arms of several races of S. araneus (Olert & Schmid, 1978; Halkka & Sцderlund, 1987). It is known there is a polymorphism for the size of NOR regions between animals belonging to the same species. It seems that the block of 18S rDNA on the o arm of the Novosibirsk race karyotype of S. araneus is too small to be revealed by FISH with the rDNA probe used. However, long cultivation of cells led to amplification of rDNA and increased the number of rDNA copies revealed by FISH. In contrast, 18S rDNA in S. granarius was revealed on the short arms of 12 acrocentric pairs, and no FISH signals for the rDNA probe were observed on chromosomes de, tu, p, o, q and r (Fig. 2D). In some chromosomes rDNA was localized in the same regions as clusters of telomeric repeats, while in the others rDNA and clusters of telomeric repeats were ordered one after another (Fig. 2 EF). An intermediate pattern in their distribution was also found. Coming back to the results obtained in the study of S. granarius large telomeres using the microdissected DNA probe, it should be noticed that according to the probe preparation it could contain various DNA sequences including telomeric, rDNA and other repeats. However, FISH with this probe gave no signal in telomeric- and 18S rDNA positive sites on S. araneus chromosomes. This means that the telomeric and rDNA are only a minor fraction of the microdissected DNA probe. It seems that pericentric regions of S. granarius acrocentrics are enriched not only with telomeric repeats and rDNA but also with other repetitive sequences. Based on comparative chromosome analysis it has been suggested that the S. granarius karyotype is similar to that ancestral for the S. araneus group (Wуjcik & Searle, 1988; Volobouev & Dutrillaux, 1991). However, reexamination of the phylogenetic relationships within the Soricidae, using methods of molecular phylogeny, revealed that S. granarius is the nearest relative to S. araneus among the species of the S. araneus group (Taberlet et al., 1994). These results allowed us to define more exactly the ancestral karyotype for species of the S. araneus group and to suggest that it includes at least four metacentric chromosomes af, bc, de, and tu. In this case, acrocentrics a, b, c and f in the S. granarius karyotype should be the result of fissions of ancestral metacentrics (Taberlet et al., 1994). The fissions apparently resulted in the formation of new telomeres on newborn acrocentrics, global reorganization of chromosomal termini and spatial nuclear organization. Telomeric repeats and rDNA should have played an important role in this process.
ACKNOWLEDGMENTS. We are deeply indebted to Dr. Vitaly Volobouev for presenting us with the cultures of primary fibroblasts of Sorex granarius. This work was partly supported by grants from RFBR (grants Nos. 05-04-48221 and 07-04-00513).

References
Azzalin C.M., Nergadze S.G. & Giolotto E. 2001. Human intrachromosomal telomeric-like repeats: sequence organization and mechanism of origin // Chromosoma. Vol.110. P.7582. Castiglia R., Gornung E. & Corti M. 2002. Cytogenetic analyses of chromosomal rearrangements in Mus minutoides/musculoides from North-West Zambia through mapping of the telomeric sequence (TTAGGG)n and banding techniques // Chromosome Research. Vol.10. P.399406. Dobigny G., Ozouf-Costaz C., Bonillo C. & Volobouev V. 2003. Evolution of rRNA gene clusters and telomeric repeats during explosive genome repatterning in Taterillus X (Rodentia, Gerbillinae) // Cytogenetic and Genome Research. Vol.103. P.94103. Halkka L. & Sцderlund V. 1987. Random NOR-activation in polymorphic and stable chromosomes of Sorex araneus L. // Hereditas. Vol.106. P.293294. Hartmann N. & Scherthan H. 2004. Characterization of ancestral chromosome fusion points in the Indian muntjac deer // Chromosoma. Vol.112. P.213220. Garagna S., Zuccotti M., Capanna E. & Redi C.A. 2002. High-resolution organization of mouse telomeric and pericentromeric DNA // Cytogenetic and Genome Research. Vol.96. P.125129. Ijdo J.W., Wells R.A., Baldini A. & Reeders S.T. 1991. Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR // Nucleic Acids Research. Vol.19. P.4780. Kilburn A.E., Shea M.J., Sargent R.G &, Wilson J.H. 2001. Insertion of a telomere repeat sequence into a mammalian gene causes chromosome instability // Molecular and Cellular Biology. Vol.21. P.126135. Malygin A.A., Graifer D.M., Zenkova M.A., Mamaev S.V. & Karpova G.G. 1992. [Affinity modification of 80S ribosomes from human placenta by derivatives of triand hexauridylates as mRNA analogs] // Molekulyarnaya Biologiya. Vol.26. P.369377 [in Russian]. Manning E.L., Crosland J., Dewey M.J. & Van Zant G. 2002. Influence of inbreeding and genetics on telomere length in mice // Mammalian Genome. Vol.13. P.234 238. Metcalfe C.J., Eldridge M.D., Toder R. & Johnston P.G. 1998. Mapping of the distribution of the telomeric sequence (TTAGGG)n in the Macropoidea (Marsupialia) by fluorescence in situ hybridization. I. The swamp wallaby, Wallabia bicolor // Chromosome Research. Vol.6. P.603610. Meyne J., Baker R.J., Hobart H.H., Hsu T.C., Ryder O.A., Ward O.G., Wiley J.E., Wurster-Hill D.H., Yates T.L. & Moyzis R.K.. 1990. Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequences in vertebrate chromosomes // Chromosoma. Vol.99. P.310. Mondello C., Pirzio L., Azzalin C.M. & Giulotto E. 2000. Instability of interstitial telomeric sequences in the human genome // Genomics. Vol.68. P.111117. Olert J. & Schmid M. 1978. Comparative analysis of karyotypes in European shrew species. I. The sibling species


Telomeric and rDNA on shrew chromosomes
Sorex araneus and S. gemellus: Q-bands, G-bands, and position of NORs // Cytogenetics and Cell Genetics. Vol.20. P.308322. Pack S.D., Borodin P.M., Serov O.L. & Searle J.B. 1993. The X-chromosome translocation in the common shrew (Sorex araneus L.): late replication in female somatic cells and pairing in male meiosis // Chromosoma. Vol.102. P.355360. Rivero M.T., Mosquera A., Goyanes V., Slijepcevic P. & Fernandez J.L. 2004. Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cell // Experimental Cell Research. Vol.295. P.161172. Rubtsov N., Senger G., Kucera H., Neumann A., Kelbova K., Junker K., Beensen V., Claussen U. 1996. Interstitial deletion of chromosome 6q: report of a case and precise definition of the breakpoints by microdissection and reverse painting // Human Genetics. Vol.97. P.705709. Ruiz-Herrera A., Garcia F., Azzalin C., Giulotto E., Ponsa M. & Garcia M. 2002. Distribution of intrachromosomal telomeric sequences (ITS) on Macaca fascicularis (Primates) chromosomes and their implication for chromosome evolution // Human Genetics. Vol.110. P.578586. Searle J.B., Fedyk S., Fredga K., Hausser J. & Volobouev V.T. 1991. Nomenclature for the chromosomes of the common shrew (Sorex araneus) // Mйmoires de la Sociйtй Vaudoise des Sciences Naturelles. Vol.19. P.13 22. Searle J.B. 1993. Chromosomal hybrid zones in eutherian mammals // Harrison R.G. (ed.). Hybrid Zones and the Evolutionary Process. New York: Oxford University Press. P.309353. Slijepcevic P. 1998. Telomeres and mechanisms of Robertsonian fusion // Chromosoma. Vol.107. P.136140.

!

Taberlet P., Fumagalli L. & Hausser J. 1994. Chromosomal versus mitochondrial DNA evolution: tracking the evolutionary history of the southwestern European populations of the Sorex araneus group (Mammalia, Insectivora) // Evolution. Vol.48. P.623636. Volobouev V.T. & Dutrillaux B. 1991. Chromosomal evolution and phylogenetic relationships of the Sorex araneus-arcticus species group // Mйmoires de la Sociйtй Vaudoise des Sciences Naturelles. Vol.19. P.131139. Wуjcik J.M. & Searle J.B. 1988. The chromosomal complement of Sorex granarius the ancestral karyotype of the common shrew (Sorex araneus)? // Heredity. Vol.61. P.225229. Wуjcik J.M., Borodin P.M., Fedyk S., Fredga K., Hausser J., Mishta A., Orlov V.N., Searle J.B., Volobouev V.T. & Zima J. 2003. The list of the chromosome races of the common shrew Sorex araneus (updated 2002) // Mammalia. Vol.67. P.169178. Zhdanova N.S., Karamisheva T.V., Minina Y., Astakhova N.M., Lansdorp P., Kammori M., Rubtsov N.B. & Searle J.B. 2005. Unusual distribution pattern of telomeric repeats in the shrews Sorex araneus and Sorex granarius // Chromosome Research. Vol.13. P.617625. Zhu L., Hathcock K.S., Hande P., Lansdorp P.M., Seldin M.F. & Hodes R.J. 1998. Telomere length regulation in mice is linked to a novel chromosome locus // Proceedings of the National Academy of Sciences of the USA. Vol.95. P.86488653. Zijlmans J.M., Martens U.M., Poon S.S., Raap A.K., Tanke H.J., Ward R.K. & Lansdorp P.M. 1997. Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats // Proceedings of the National Academy of Sciences of the USA. Vol.94. P.74237428.