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CONTAINER FOR CULTIVATING BLOOD FOR CHROMOSOME STUDIES
1362 " (3) In the 18 deteriorations of existing disability ", despite small numbers, the- favourable corticotrophin response is significantly demonstrated. It is not demonstrated significantly amongst the remainder. TJrnv iBATT T "’D HENRY MILLER
Royal Victoria Infirmary, Newcastle upon Tyne.
TABLE II-DEFINITION AND DEGREE OF HETEROPYKNOSIS OF THE SATELLITE REGION IN A SPECIFIC CHROMOSOME, IDENTIFIABLE ON ACCOUNT OF A LARGE SATELLITE
(50 cells in
each
case)
D. J. NEWELL ALAN RIDLEY.
OBSERVATIONS ON THE SATELLITED HUMAN CHROMOSOMES SIR,-In their comments on our articlewith this title Dr. Petersen and Dr. Therkelsen2 question the validity of our definition of a satellited chromosome, and imply that "
distinct chromatin knobs " and " negative heteropyknosis at the ends of the chromosome arms " need not, as we affirm, be manifestations of the same phenomenon. Our observations indicate that satellited regions vary in their morphological appearance and degree of heteropyknosis under conditions at present only partially apparent, and lead to the conclusion that all chromosomes in groups IV (13-15) and VII (21 and 22) are potentially satellited. Additional evidence for this may be found in the answers to the three questions posed by Dr. Petersen and Dr. Therkelsen. The first question inquires about the proportion of satellited chromosomes which appear as " distinct chromatin knobs " rather than areas of negative heteropyknosis. As shown in table I, the proportion of " distinct knobs " found in the three
satellite association. In passing we might add that we have heard confirmation of the phenomenon of satellite association from many investigators. It appears also that this phenomenon is not confined to man, as one of us has made similar observations in the chimpanzee (M. A. F.-S., unpublished), and we hear that it has been seen in swine cells (Dr. Frank H. Ruddle,
personal communication). We have noted elsewhere that secondary constrictions in non-satellited chromosomes either appear as regions of negative heteropyknosis or as isopyknotic constrictions, or merely as regions associated with satellited chromosomes.3 In their variable degree of heteropyknosis they are very similar to the satellite regions in the chromosomes of groups IV and VII. Division of Medical Genetics Department of Medicine,
Johns Hopkins University School of Medicine, Baltimore, Maryland.
TABLE I-TYPE OF SATELLITE REGION PRESENT IN UNSELECTED GROUP IV AND GROUP VII CHROMOSOMES IN 150 CELLS FROM THREE INDIVIDUALS
MALCOLM A. FERGUSON-SMITH STANLEY D. HANDMAKER.
CONTAINER FOR CULTIVATING BLOOD FOR CHROMOSOME STUDIES
step in the cultivation of white blood-cells by the technique of Moorhead et a1.4 is the separation of the red cells from the plasma containing the white cells. When the amount of supernatant plasma is small it is important to make the most of it and avoid contamination with red cell.
SIR,-An important
pairs of group-iv chromosomes is not very different from the proportion found in the two pairs of group-vn chromosomes, in each group the satellite appearing as a " negative heteropyknotic region " approximately 5-6 times more frequently than as distinct knobs ". These figures are consistent with all chromosomes in both groups being potentially satellited. In answer to the second question, we have never seen 10 chromosomes with distinct chromatin knobs in any one diploid "
cell. Needless to say, had we done so, none of the tedious counts recorded in tables I and II of our article would have been necessary. One photograph would have been sufficient. The answer to question 3 is yes. We have on four occasions noted cells in which five group-iv chromosomes have satellites which appear as "secondary constriction with distinct chromatin knobs projected from the arms on slender stalks ". Table II is presented as evidence that one satellited region may vary in morphological appearance and show different degrees of heteropyknosis from one cell to another in a particular individual. One of us (S. D. H.) has examined material from three individuals in which a satellited chromosome in group iv, or group VII, or both groups, can be readily distinguished from other chromosomes in the same group because of the differential size of the satellite. In each case this satellite
region can be identified either as larger isopyknotic knobs, or as larger negatively heteropyknotic knobs, or as larger areas of negative heteropyknosis. Observations on satellite association also tend to confirm that all groups IV and VII chromosomes are potentially satellited, for we have several times observed a cell in which all group-vn chromosomes or all group-iv chromosomes are involved in 1. Lancet, 1961, i, 638. 2. ibid. p. 1229.
I have found it useful in these
cases to use
glass container with a constriction in the middle (see figure). The heparinised blood is put directly in this container, and after a
centrifugation the interface between plasma and red cells is at the narrow part of the container; this makes it easier to obtain the maximum amount of red-cell-free Dlasma. Central Hospital, Eskilstuna, KURT Sweden.
KAIJSER.
ACRIDINE ORANGE AS A CHROMOSOME STAIN
SIR,-One of the problems associated with the interpretation of small chromosomal fragments in the human cell cultures is the presence of non-chromosomal debris in the microscopic fields. Two of us (L. M. S. and T. V.) have developed a method for staining human chromosomes with acridine orange, which offers the advantages of good contrast and the absence of stained artefacts. Although the method will be published in detail elsewhere, your readers may care to have a brief account of it. Chromosome preparations were made from leucocytes cultured by the method of Moorhead et a1.4 They were allowed on non-fluorescent slides. The chromosomes were the slides with absolute methyl alcohol, and the slides were progressively transferred through diminishing concentrations of ethyl alcohol and finally to distilled water. They were then placed in McIlvaine’s buffer, pH 6-4, stained with 0-01%
to
air-dry
fixed
3. 4.
to
Ferguson-Smith, M. A., Handmaker, S. D. To be published. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, Hungerford, D. A. Exp. Cell Res. 1960, 20, 613.
D.
M.,
Royal Victoria Infirmary, Newcastle upon Tyne.
TABLE II-DEFINITION AND DEGREE OF HETEROPYKNOSIS OF THE SATELLITE REGION IN A SPECIFIC CHROMOSOME, IDENTIFIABLE ON ACCOUNT OF A LARGE SATELLITE
(50 cells in
each
case)
D. J. NEWELL ALAN RIDLEY.
OBSERVATIONS ON THE SATELLITED HUMAN CHROMOSOMES SIR,-In their comments on our articlewith this title Dr. Petersen and Dr. Therkelsen2 question the validity of our definition of a satellited chromosome, and imply that "
distinct chromatin knobs " and " negative heteropyknosis at the ends of the chromosome arms " need not, as we affirm, be manifestations of the same phenomenon. Our observations indicate that satellited regions vary in their morphological appearance and degree of heteropyknosis under conditions at present only partially apparent, and lead to the conclusion that all chromosomes in groups IV (13-15) and VII (21 and 22) are potentially satellited. Additional evidence for this may be found in the answers to the three questions posed by Dr. Petersen and Dr. Therkelsen. The first question inquires about the proportion of satellited chromosomes which appear as " distinct chromatin knobs " rather than areas of negative heteropyknosis. As shown in table I, the proportion of " distinct knobs " found in the three
satellite association. In passing we might add that we have heard confirmation of the phenomenon of satellite association from many investigators. It appears also that this phenomenon is not confined to man, as one of us has made similar observations in the chimpanzee (M. A. F.-S., unpublished), and we hear that it has been seen in swine cells (Dr. Frank H. Ruddle,
personal communication). We have noted elsewhere that secondary constrictions in non-satellited chromosomes either appear as regions of negative heteropyknosis or as isopyknotic constrictions, or merely as regions associated with satellited chromosomes.3 In their variable degree of heteropyknosis they are very similar to the satellite regions in the chromosomes of groups IV and VII. Division of Medical Genetics Department of Medicine,
Johns Hopkins University School of Medicine, Baltimore, Maryland.
TABLE I-TYPE OF SATELLITE REGION PRESENT IN UNSELECTED GROUP IV AND GROUP VII CHROMOSOMES IN 150 CELLS FROM THREE INDIVIDUALS
MALCOLM A. FERGUSON-SMITH STANLEY D. HANDMAKER.
CONTAINER FOR CULTIVATING BLOOD FOR CHROMOSOME STUDIES
step in the cultivation of white blood-cells by the technique of Moorhead et a1.4 is the separation of the red cells from the plasma containing the white cells. When the amount of supernatant plasma is small it is important to make the most of it and avoid contamination with red cell.
SIR,-An important
pairs of group-iv chromosomes is not very different from the proportion found in the two pairs of group-vn chromosomes, in each group the satellite appearing as a " negative heteropyknotic region " approximately 5-6 times more frequently than as distinct knobs ". These figures are consistent with all chromosomes in both groups being potentially satellited. In answer to the second question, we have never seen 10 chromosomes with distinct chromatin knobs in any one diploid "
cell. Needless to say, had we done so, none of the tedious counts recorded in tables I and II of our article would have been necessary. One photograph would have been sufficient. The answer to question 3 is yes. We have on four occasions noted cells in which five group-iv chromosomes have satellites which appear as "secondary constriction with distinct chromatin knobs projected from the arms on slender stalks ". Table II is presented as evidence that one satellited region may vary in morphological appearance and show different degrees of heteropyknosis from one cell to another in a particular individual. One of us (S. D. H.) has examined material from three individuals in which a satellited chromosome in group iv, or group VII, or both groups, can be readily distinguished from other chromosomes in the same group because of the differential size of the satellite. In each case this satellite
region can be identified either as larger isopyknotic knobs, or as larger negatively heteropyknotic knobs, or as larger areas of negative heteropyknosis. Observations on satellite association also tend to confirm that all groups IV and VII chromosomes are potentially satellited, for we have several times observed a cell in which all group-vn chromosomes or all group-iv chromosomes are involved in 1. Lancet, 1961, i, 638. 2. ibid. p. 1229.
I have found it useful in these
cases to use
glass container with a constriction in the middle (see figure). The heparinised blood is put directly in this container, and after a
centrifugation the interface between plasma and red cells is at the narrow part of the container; this makes it easier to obtain the maximum amount of red-cell-free Dlasma. Central Hospital, Eskilstuna, KURT Sweden.
KAIJSER.
ACRIDINE ORANGE AS A CHROMOSOME STAIN
SIR,-One of the problems associated with the interpretation of small chromosomal fragments in the human cell cultures is the presence of non-chromosomal debris in the microscopic fields. Two of us (L. M. S. and T. V.) have developed a method for staining human chromosomes with acridine orange, which offers the advantages of good contrast and the absence of stained artefacts. Although the method will be published in detail elsewhere, your readers may care to have a brief account of it. Chromosome preparations were made from leucocytes cultured by the method of Moorhead et a1.4 They were allowed on non-fluorescent slides. The chromosomes were the slides with absolute methyl alcohol, and the slides were progressively transferred through diminishing concentrations of ethyl alcohol and finally to distilled water. They were then placed in McIlvaine’s buffer, pH 6-4, stained with 0-01%
to
air-dry
fixed
3. 4.
to
Ferguson-Smith, M. A., Handmaker, S. D. To be published. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, Hungerford, D. A. Exp. Cell Res. 1960, 20, 613.
D.
M.,