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First published online 2 November 2004
doi: 10.1242/jcs.01503

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DNA replication licensing in somatic and germ cells

Kathryn Leigh Eward^1,*, Ellen C. Obermann^2,*, S. Shreeram³, Marco Loddo², Thomas Fanshawe⁴, Craig Williams¹, Hyo-Il Jung¹, A. Toby Prevost⁵, J. Julian Blow³, Kai Stoeber^1,2, and Gareth H. Williams^1,2

¹ Wolfson Institute for Biomedical Research, University College London, The Cruciform Building, Gower Street, London, WC1E 6BT, UK
² Department of Histopathology, University College London, Rockefeller Building, University Street, London, WC1E 6JJ, UK
³ Welcome Trust Biocentre, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
⁴ Centre for Applied Medical Statistics and General Practice and Primary Care Research Unit, Department of Public Health and Primary Care, University of Cambridge, Forvie Site, Robinson Way, Cambridge, CB2 2SR, UK
⁵ Medical Research Council Biostatistics Unit, Institute of Public Health, Forvie Site, Robinson Way, Cambridge, CB2 2SR, UK

View larger version (46K): [in a new window]   Fig. 1. Generation and characterisation of anti-Geminin antibodies. (A) Coomassie staining of purified bacterially expressed His6-Geminin. (B) Immunoblots of recombinant Geminin and human cell lysates with affinity-purified anti-Geminin antibodies G94 and G95. Antibodies G94 and G95 detected a single 33kDa protein in total cell lysates from asynchronous Jurkat, HeLa, MOLT-4 and HL-60 cells, and recognised nanogram quantities of recombinant Geminin (35 kDa) whereas preimmune (PI) sera did not. (C) Immunoprecipitation (IP) of HeLa S phase cell lysates with antibody G94 followed by immunoblotting (IB) with G95 shows the 33 kDa protein in the immunoprecipitate whereas immunoprecipitation with preimmune (PI) sera did not. (D) Bivariate flow cytometry of asynchronous MOLT-4 cells with antibody G95 alone, or G95 plus recombinant Geminin. Note that cells in G1 (2C) are Geminin-negative (depicted as area within the trapezoidal region) whereas cells in S or G2/M are positive (area outside the trapezoidal region). Preincubation of antibody G95 with recombinant Geminin blocked detection of this cell cycle specific expression.

View larger version (50K): [in a new window]   Fig. 2. Expression profiles of origin licensing factors during the mitotic cycle of human synchronous cells. (A) Schematic of membrane elution methodology. Asynchronously proliferating MOLT-4 cells are immobilised on surfaces such that cell division results in release of one daughter cell into the effluent while the other daughter cell remains surface-bound. Newborn (early G1 phase) cells are continuously released in the effluent and grow synchronously without evidence of disturbance. (B) FACS DNA profiles of synchronously proliferating MOLT-4 cells at 3-hour intervals. (C) (Top) Cell-cycle-phase distributions as determined by DNA profiles in B. Immunoblots of origin licensing factors and control proteins in total cell lysates from equivalent numbers of synchronously proliferating MOLT-4 cells at the indicated times. (D) In situ staining of Geminin (G95) in asynchronous, early G1-phase and S/G2 phase MOLT-4 cells. Synchronous cells were obtained using the membrane elution method.

View larger version (89K): [in a new window]   Fig. 3. Ki67, Cdc6, Cdt1, Mcm2 and Geminin protein expression in human colonic mucosa (original magnification x144). The schematic drawing of a colonic crypt (cross section; top left) shows the basal proliferative compartment (BC; white star) containing stem-transit progenitor cells and the luminal compartment (LC; black star) containing differentiating goblet and enteroendocrine cells. A large proportion of cells in the BC express Ki67. The majority of basal cells and a subset in the LC express Mcm2 and Cdt1. Geminin expression is restricted to a subset of cells in the BC. Cdc6 expression is restricted to basal proliferating cells. Note the absence of origin licensing factors at the base of the crypts (putative stem cell compartment).

View larger version (107K): [in a new window]   Fig. 4. Ki67, Cdc6, Cdt1, Mcm2, and Geminin protein expression in human seminiferous tubules (original magnification x400). The schematic drawing (Top left) of a seminiferous tubule (cross section) shows the basal compartment (BC) containing stem cell spermatogonia directly in contact with the basement membrane (arrow 1). The differentiating luminal compartment (LC) contains germ cells in meiosis and postmeiotic phases. Maturing spermatogonia enter meiosis and become primary spermatocytes (arrow 2). Primary spermatocytes reside in a prolonged prophase of MI. Completion of MI and MII produces haploid early spermatids (arrow 3) with further differentiation giving rise to late spermatids (arrow 4). Ki67 expression is restricted to the BC. The majority of primary spermatocytes and early spermatids express Mcm2 including a subpopulation of spermatogonia. High levels of Geminin are detected in the LC. The majority of primary spermatocytes show strong immunostaining for Geminin. A small number of spermatogonia express Geminin (arrowhead). Expression of Cdc6 and Cdt1 is found in significant numbers of primary spermatocytes (thin arrows) and a subpopulation of spermatogonia (thick arrows).

View larger version (32K): [in a new window]   Fig. 5. The proliferation-differentiation switch in colon and loss of DNA replication competence are coupled to downregulation of Ki67, Cdc6, Cdt1 and Geminin. In testis, the proliferation-differentiation switch and loss of DNA replication competence are also coupled to downregulation of Ki67. However, in contrast to somatic cells, early stages of germ cell differentiation and entry into meiosis are associated with high levels of Cdc6, Cdt1, Mcm2 and Geminin. Late stages of differentiation in somatic and germ cell self-renewing systems are linked to downregulation of Cdc6, Cdt1, Mcm2 and Geminin. Note that in contrast to Ki67, Cdc6, Cdt1 and Geminin, downregulation of Mcm2 is coupled to the final stages of the differentiation programme. BC, basal compartment; LC, luminal compartment; TDC, terminally different compartment; SG, spermatogonia; SC, primary spermatocytes; EST, early spermatids and late spermatids (LST)

View larger version (128K): [in a new window]   Fig. 6. Ki67, Cdc6, Cdt1, Mcm2 and Geminin protein expression in human ovary (original magnification x120). The schematic drawing of adult ovary (cross section; top left) shows that the predominant follicles are primordial follicles (white arrow) composed of primary oocytes arrested in the prophase of MI and surrounded by flattened follicular cells (black thin arrow). Primordial follicles mature into Graafian follicles following a period of follicular growth. Follicular growth involves proliferation of follicular cells and increase in size of oocytes to form primary follicles (white arrowhead). Primary oocytes do not express Ki67 contrasting with expression in follicular cells of primary follicles. Virtually all primary oocytes express Cdt1 and Mcm2 proteins. Geminin is found in a proportion of proliferating follicular cells, but is not expressed in primary oocytes. Cdc6 is detected in proliferating follicular cells of primary and secondary follicles (inset; arrowhead) but not in primary oocytes.

View larger version (12K): [in a new window]   Fig. 7. Labelling indices for primary spermatocytes and primary oocytes.

View larger version (33K): [in a new window]   Fig. 8. Analysis of geminin splice variants in human cell lines and tissues and detection of Geminin and Cdc6 proteins in testis but not ovary. (A) Detection of two geminin isoforms after RT-PCR of total RNA in cell lines using geminin-specific primers. In the first lane a control PCR without DNA is shown. Actin was amplified as a positive control for each sample. (B) Detection of geminin isoforms after RT-PCR of total RNA in human tissues as described above. Actin was amplified as a positive control for each sample and a control PCR without actin was negative as above (data not shown). (C) Amplification plot resulting from quantitative real-time RT-PCR of Geminin mRNA splice variant GemE and endogenous control 18s rRNA (inset) in RNA from human testis, ovary, colon and cervix. Dashed lines represent RNA from human testis whereas solid lines represent RNA from human ovary, colon and cervix. (D) Immunoblots of Geminin in human testis and ovary tissue lysates (75 µg each/lane) with affinity-purified antibodies G94 and G95 alone, or after preincubation with recombinant Geminin. A band of approximately 20 kDa (arrow) was also detected with both G94 and G95. This interaction was blocked after preincubation of the antibodies G94 and G95 with recombinant Geminin. (E) Immunoblots of total cell lysates from testis and ovary with an anti-Cdc6 antibody.

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