Section 16 — Human Cytogenetics 



The DNA content of cell nuclei and individual 

 chromosomes was studied. The cells and chromo- 

 somes were stained by the Feulgen method and 

 the DNA measured by a new microspectro- 

 photometric method based on photographic 

 colorimetry. 



A photomicrograph from the stained object is 

 taken with monochromatic light. The negative 

 is enlarged and by a special development a blue 

 colored print is made. In this print the pictures of 

 the object to be measured are cut out, the blue 

 dye is extracted and the extinction of this solution 

 is determined with a colorimeter. 



As is postulated in the constancy hypothesis, 

 all diploid cells in an organism should have the 

 same DNA content. When measuring a number 

 of cells, a certain variation in the results is found. 

 Analysis of variance was done to separate the 

 variation in the object from the random measur- 

 ing error. In this way we established the accuracy 

 of the method and tested the constancy hypo- 

 thesis. 



We found a high constancy of DNA content. 

 The measured ratio between the DNA content 

 of diploid and tetraploid cells also fulfilled the 

 theoretical requirements. 



Photographic colorimetry has the advantage 

 that objects of very different forms can be 

 measured by cutting out the pictures from the 

 photograph. This fact and the results with inter- 

 phase nuclei led us to use the method for the 

 determination of the DNA content of individual 

 human chromosomes. These results will be 

 discussed. 



16.8. Further Characterization of the Chromosomal 

 Complement of Man Through Autoradiography. 



James L. German (New York City, U.S.A.). 



In addition to their structural features, the 

 sequence in which chromosomes complete DNA 

 replication provides further characterization. This 

 sequence has been partially elucidated through 

 autoradiography of metaphase chromosomes 

 in which the DNA replicated during the terminal 

 1-3 hours of the S-period had been labeled with 

 H 3 -thymidine. 



Though variability and homologue asynchrony 

 are prominent in nucleated blood cells in vitro, 

 intra-group patterns in Groups 4-5, 13-15, 16-18, 

 and Y-21-22 serve to distinguish members of the 

 group. Two in Group 4-5 are among the latest 

 of the complement to complete replication, 



while in the other two there is early completion 

 in the long arms. In Group 13-15 two are very 

 late, while two are among the earliest of the 

 complement to cease the uptake of thymidine. 

 Pair No. 17 completes replication very early, 

 pair No. 18 late. Of chromosomes of Group 

 Y-21-22, in most male cells the Y is last; two of 

 the group complete replication very early. 



The late replicating X of cells derived from the 

 normal female is clearly distinguished from 

 others of Group 6-X-12. Its characteristics 

 include: 51 per mille of the total length of the 

 haploid complement; arm ratio of 1.75; extreme 

 peripheral location in 33 per cent of c-metapha- 

 ses (expected in 32 per cent); secondary con- 

 striction in the long arm in 3 per cent of cells; 

 length in relation to the average length of a 



average 4-5 



Group 4-5 chromosome ( )= 1.25; 



inordinate shortening in relation to chromosomes 

 of Group 4-5 in cells having had exposure to 

 colcemide for 3 hours. 



16.9. Autoradiographic Studies of Normal and 

 Abnormal Human Karyotypes. Werner Schmid 

 (Houston, U.S.A.). 



Chromosomes replicate segments of their 

 deoxyribonucleic acid (DNA) strands in non- 

 random sequences. The use of tritiated thymi- 

 dine and autoradiography permit the study of 

 these replication sequences in metaphase chro- 

 mosomes, a technique which was introduced 

 into cytogenetics by Taylor, Woods and Hughes 

 in 1957 (!). 



In short-term cultures of human leukocytes 

 and in embryonic fibroblast cultures, continuous 

 labeling with tritiated thymidine (specific activity 

 1.9 C/mmol) at a concentration of 1 uc/ml 

 medium, was begun 6 hours preceding fixation 

 of the cells in metaphase. Colcemid was added 

 for the last two hours, collecting cells in meta- 

 phase from different terminal DNA replication 

 stages. The chromosomes were photographed 

 before and after exposure (4-6 days) to auto- 

 radiographic film. The often stepwise sequences 

 in which DNA replication is completed in 

 individual chromosome pairs will be demon- 

 strated. Homologous chromosomes go through 

 the same final replication sequences, usually 

 synchronously, sometimes slightly out of phase. 

 Five very marked, late replicating regions in 

 chromosome pairs Nos. 1,4, 9, 16 and in the Y, 

 coincide with secondary constrictions which, by 

 means of special techniques, have been demon- 

 strated in these chromosomes by Saksela and 

 Moorhead( 2 ). So far, no significant differences 



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