Cytological characterisation of heterochromatin in mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana (Diptera: Calliphoridae) D. G. BEDO Division of Entomology, Common wealth Scientific and Industrial Research Organisation, G. P. 0. Box 1700, Canberra City, Australian Capital Territory 2601, Australia Corresponding Editor: P. B. Moens
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Received January 3, 1991 Accepted April 25, 1991 BEDO,D. G. 1991. Cytological characterisation of heterochromatin in mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana (Diptera: Calliphoridae). Genome, 34: 631-637. Mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana, were studied using C-banding and quinacrine and counterstain-enhanced fluorescence techniques. The five autosomes in the karyotype are evenly graded in size, with somewhat variable arm ratios. Distinguishing all autosomes on these features alone can be difficult. C-banding produces small centromeric bands in the autosomes, whereas the much longer X and Y chromosomes have extensive dark C-band blocks with intermediate background staining. Most bright fluorescence occurs in the sex chromosomes, particularly the X chromosome, which has remarkable banding detail. Band resolution is greatly increased in mitotic metaphase cells from embryos. Quinacrine staining of mitotic chromosomes produces bright fluorescence at the centromere regions of chromosomes 2, 3, and 4, assisting in their identification. Meiotic chromosomes have distinctly reduced brightness and resolution of fluorescent bands and show marked chromatid asynapsis in the brighter regions of the sex chromosomes. Fluorochromes staining A . T-rich DNA (quanacrine and 4,6-diamidino-2-phenylindole (DAPI)) produce bright staining in a large proportion of the sex chromosomes. By contrast chromomycin, which binds preferentially to G .C-rich DNA, stains a much smaller proportion of the sex chromosomes than expected from reciprocal staining. Together with the asynapsis data this indicates that much of the heterochromatin in the sex chromosomes has unusual structural properties. Key words: Chrysomya bezziana, screwworm, karyotype, C-banding, fluorescence, heterochromatin. BEDO,D. G. 1991. Cytological characterisation of heterochromatin in mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana (Diptera: Calliphoridae). Genome, 34 : 631-637. Des chromosomes mitotiques et meiotiques de la mouche (( fil de vis n, Chrysomya bezziana, ont ete etudies au moyen de techniques de coloration des bandes C et de coloration a la quinacrine, avec fluorescence rehaussee par des contrecolorants. Les cinq autosomes du caryotype sont egaux en dimension, avec quelques variations dans les ratios de la longueur des bras. I1 peut donc s'averer difficile de distinguer les autosomes les uns des autres par ces caracteres. La coloration des bandes C a revele la presence de petites bandes centromeriques chez les autosomes, alors que les chromosomes X et Y, beaucoup plus longs, possedaient des blocs extensifs et fonces de bandes C sur un fond intermediaire de coloration. La presque totalite de la fluorescence vive s'est manifestee chez les chromosomes du sexe, particulierement le chromosome X, dont le detail des bandes a ete remarquable. La resolution des bandes a ete fortement augmentee chez les cellules d'embryons en metaphase mitotique. La coloration a la quinacrine des chromosomes mitotiques a produit une fluorescence brillante dans les regions centromeriques des chromosomes 2, 3 et 4, ce qui a facilite leur identification. La luminosite de la fluorescence a ete nettement diminuee chez les chromosomes meiotiques, de meme que la resolution des bandes, mais elle a revele une asynapsis marquee des chromatides dans les regions de plus vive fluorescence des chromosomes du sexe. Les fluorochromes (quinacrine et DAPI), qui ont co!ore I'ADN riche en A . T, ont produit une coloration lumineuse dans une large proportion des chromosomes du sexe. A l'oppose, la chromomycine, qui se lie preferablement a I'ADN riche en G .C, a colore une proportion beaucoup plus faible des chromosomes du sexe que ce qui etait attendu d'une coloration reciproque. Ces donnees, ajoutees a celles de l'asynapsis, indiquent qu'une large proportion de l'heterochromatine des chromosomes du sexe possede des proprietes structurales inhabituelles. Mots clks : Chrysomya bezziana, mouche (( fil de vis n, caryotype, bandes C, fluorescence, heterochromatine. [Traduit par la redaction]
Introduction The Old World screwworm fly, Chrysomya bezziana, is a serious economic and health menace extending from South Africa through the Middle East and India to southeast Asia and New Guinea. A wide range of host animals and humans is attacked by this obligate parasite (Spradbery and Vanniasingham 1980). Although no populations have been established in Australia, its introduction presents a major economic threat to livestock industries. This, together with recent discoveries of live adults on ships originating from the Middle East and aircraft from India (Rajapaksa and Spradbery 1989), has provided impetus for development of a control strategy. Currently this is fulfilled by research into Printed in Canada / Imprime au Canada
the sterile insect release method (SIRM) (Knipling 1955) based in Port Moresby, Papua New Guinea. The New World screwworm, Cochliomyia homonivorax, is a similar economic and health problem currently the subject of a major SIRM control program in the United States and Mexico. Cochliomyia homonivorax has been the subject of speculation concerning genetic differentiation between natural populations (Lachance and Whitten 1986; Richardson et al. 1982). Such differentiation, if it existed in C. bezziana, would have great practical impact in the application of genetic control. In response to its introduction into Australia from different geographic regions, the cytogenetic study of C. bezziana has been intensified. A stan-
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632
GENOME, VOL. 34, 1991
dard polytene chromosome map has been compiled (D.G. Bedo, in preparation) and a comparison of banding patterns in populations throughout its range is in progress. Polytene chromosome analysis allows detailed study of fully replicated autosomal regions but fails to deal with underreplicated heterochromatin. Mitotic chromosomes must be used to study heterochromatin underreplicated in polytene cells. Measurement of chromosome lengths and arm ratios can show gross variation between populations, for example, the sex chromosomes of Co. homonivorax (LaChance and Whitten 1986). Finer resolution can be achieved by applying chromosome banding techniques to reveal cryptic variation within heterochromatin. Quinacrine staining and counterstain-enhanced fluorescence, which allow chromatin with a particular DNA content to be differentiated (Schweizer 1981, 1983), are particularly useful. For example, numerous bands revealed by fluorescence staining allow detailed cytogenetic analysis of the sex chromosomes of Drosophila melanogaster (Gatti and Pimpinelli 1983; Pimpinelli et al. 1985) and the Mediterranean fruit fly, Ceratitis capitata (Bedo 1989). Quinacrine banding applied to Co. homonivorax (McInnis 1981; McInnis et al. 1983; Richardson et al. 1982) detected sex chromosome variation in populations targeted for control by sterile insect release. Hybridization studies indicated that these differences represent polymorphism in natural populations (LaChance and Whitten 1986). This report details the characteristics of heterochromatic regions in mitotic and meiotic chromosomes of C. bezziana, using C-banding, quinacrine fluorescence, and counterstain-enhanced fluorescence. Materials and methods Flies Because the importation of live C. bezziana into Australia is prohibited, all mitotic and meiotic chromosome material was prepared during a visit to the New Guinea laboratory where mass-reared colonies are maintained. Chromosome preparation Air-dried mitotic chromosome preparations were made from third-instar larval brains as described by Bedo (1980). The same technique was modified to make air-dried preparations of whole embryos. Eggs were harvested approximately 3 h after laying and dechorionated by rolling them on the sticky side of a strip of adhesive tape. Dechorionated eggs were then placed in a drop of freshly mixed acetic alcohol and pierced with a sharp needle to allow penetration of the fixative. After 5 min the fixed eggs were transferred to a drop of 65% acetic acid on a slide and macerated with dissecting needles. The remaining steps are the same as those described earlier (Bedo 1980) for larval brain preparations. Chromosome measurements Chromosome arm measurements were made with vernier callipers from photographic prints of well-spread metaphase cells. The karyotype was visually ranked according to length before measurement. Chromosome arm ratios of all chromosomes were calculated and subjected to pairwise t-tests to compare centromere positions of all chromosomes. To quantify relative chromosome lengths, the percent of total haploid autosomal complement length (%TACL) for each autosome was calculated. Measurements of autosomes were also tested with paired t-tests for relative length differences. By only using the autosomes male and female cells could be pooled. The sex chromosomes were treated separately by calculating the length ratios of the X chromosome to the Y chromosome in male cells and the X chromosome to chromosome 2 in pooled male and female cells.
FIG. 1. Idiogram representing the karyotype of C. bezziana. The centromeres are indicated by constrictions. Nucleolar organiser regions in XL and YL are shown by gaps near the distal ends of the arms. Chromosome lengths and arm ratios are drawn in proportion.
Banding procedures C-banding and quinacrine staining were carried out as described by Bedo (1980). The counterstain-enhanced tristaining fluorescence method with chromomycin A! (CMA), 4,6-diamidino-2phenylindole (DAPI), and distamycin (Schweizer 1980, 1981, 1983) was also used. This allows CMA and distamycin-DAPI (DADAPI) staining to be observed in the same chromosome by simply changing excitation and barrier filter combinations. For sequential C-banding of tristained chromosomes, slides were destained in acetic ethanol for 24 h before applying C-banding procedures with reduced Ba(OH)2 pretreatment or staining in 2% Giemsa with no pretreatment (Bedo 1989).
Results Chrysomya bezziana has an XX/XY sex-chromosome system, designated here, using Drosophila conventions, as the first pair of the karyotype, and five pairs of autosomes numbered as pairs 2-6 (Figs. 1 and 2). Measurements (Table 1) show chromosomes 2 and 5 to be acrocentric, with chromosomes 3, 4, and 6 more metacentric in arm ratio. The autosomes form an evenly graded series in relative length, with chromosomes 3 and 4 being the most similar. Chromosome 3 is somewhat more acrocentric than chromosome 4, with a significantly different arm ratio (Tables 1 and 2). Chromosomes 3 and 4 are often difficult to distinguish visually, but as shown below, the application of quinacrine banding simplifies their identification. At over twice the length of the largest autosome pair, the X and Y chromosomes dominate the karyotype (Figs. 1 and 2; Table 3). A well-expressed secondary constriction occurs near the end of the Y chromosome long arm (YL) with an additional secondary constriction of variable expression more proximally in YL. The X chromosome has a small satellite at the end of the long arm (XL), which is easily seen only in extended prophase or early metaphase chromosomes. The prominent YL secondary constriction and the constriction separating the XL satellite are nucleolar organiser regions (NORs) containing ribosomal genes (D. G. Bedo, submitted).
TABLE1. Arm ratios ( + SD) and %TACL ( + SD) of 82 autosome complements from 58 mitotic metaphase cells and 9 second meiotic metaphase cells
TABLE2. Paired t-tests for differences between arm ratios and lengths of autosomes of the cells used in Table 1 Chromosome No. Chromosome No.
Chromosome No.
Arm ratio %TACL
1.83
1.43
( + 0.48)
(+ 0.03)
24.7
21.5
(+ 0.01)
(+ 0.02)
1.29 (k0.06) 20.2 ( + 0.03)
1.97
1.22
(k0.13)
(k0.05)
18.0
16.6
(k0.01)
(+ 0.01)
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TABLE3. Measurements (mean + SD) of X and Y chromosomes from cells used in Table 1 Arm ratio X*
Yt
X/Y ratio7
X/2 ratio*
*For these measurements averaged X chromosomes and second autosomes from 13 female cells were pooled with X chromosomes and averaged second autosome pairs from 45 male cells from Table 1. tForty-five X and Y chromosome pairs from the male mitotic metaphase cells were used.
The number of bands resolved in the sex chromosomes by all the techniques used varies greatly with the degree of chromosome contraction. Extended late prophase and early metaphase chromosomes are particularly suitable, showing more bands than contracted metaphase figures, in which banding groups tend to fuse into blocks. Embryo chromosomes are typically far less contracted than mitotic karyotypes and show the greatest resolution of bands.
C-banding Dark C-banding is produced mainly in the sex chromosomes (Figs. 2 and 10). Autosomal staining is limited to small centromeric bands, which do not consistently appear on every chromosome in a cell but are found on all autosomes in a sample of cells. This is probably a result of variations in the staining procedure sometimes resulting in overtreatment of these fine bands. The Y chromosome is darkly stained in the short arm (YS) and the proximal half of the long arm (YL). A region of reduced stain intensity occurs in the distal half of YL. This has intermediate "grey" staining, which is darker than the general pale staining of the autosomes. A distinct dark band obscures the secondary constriction region containing the Y chromosome NOR site near the end of the arm. An additional constriction, which is usually ill defined, lies close to the proximal end of the grey region. In the X chromosome, staining differentiation is more complex, with bands of varying intensity on a background of light- to medium-strength staining (Figs. 2 and 10). As in the Y chromosome, the distal half of XL has generally paler staining, with a dark band obscuring the NOR site in the secondary constriction at the tip of the arm. Faint bands may be distinguished in the distal grey region of XL, but these are not consistent. A large, intense C-band block marks the proximal end of the grey region. The centromere lies in an area containing four indistinct bands. The base of XS
2
3
4
5
6
NOTE:Arm ratios are compared above the diagonal; length comparisons are shown below the diagonal. *p
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Received January 3, 1991 Accepted April 25, 1991 BEDO,D. G. 1991. Cytological characterisation of heterochromatin in mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana (Diptera: Calliphoridae). Genome, 34: 631-637. Mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana, were studied using C-banding and quinacrine and counterstain-enhanced fluorescence techniques. The five autosomes in the karyotype are evenly graded in size, with somewhat variable arm ratios. Distinguishing all autosomes on these features alone can be difficult. C-banding produces small centromeric bands in the autosomes, whereas the much longer X and Y chromosomes have extensive dark C-band blocks with intermediate background staining. Most bright fluorescence occurs in the sex chromosomes, particularly the X chromosome, which has remarkable banding detail. Band resolution is greatly increased in mitotic metaphase cells from embryos. Quinacrine staining of mitotic chromosomes produces bright fluorescence at the centromere regions of chromosomes 2, 3, and 4, assisting in their identification. Meiotic chromosomes have distinctly reduced brightness and resolution of fluorescent bands and show marked chromatid asynapsis in the brighter regions of the sex chromosomes. Fluorochromes staining A . T-rich DNA (quanacrine and 4,6-diamidino-2-phenylindole (DAPI)) produce bright staining in a large proportion of the sex chromosomes. By contrast chromomycin, which binds preferentially to G .C-rich DNA, stains a much smaller proportion of the sex chromosomes than expected from reciprocal staining. Together with the asynapsis data this indicates that much of the heterochromatin in the sex chromosomes has unusual structural properties. Key words: Chrysomya bezziana, screwworm, karyotype, C-banding, fluorescence, heterochromatin. BEDO,D. G. 1991. Cytological characterisation of heterochromatin in mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana (Diptera: Calliphoridae). Genome, 34 : 631-637. Des chromosomes mitotiques et meiotiques de la mouche (( fil de vis n, Chrysomya bezziana, ont ete etudies au moyen de techniques de coloration des bandes C et de coloration a la quinacrine, avec fluorescence rehaussee par des contrecolorants. Les cinq autosomes du caryotype sont egaux en dimension, avec quelques variations dans les ratios de la longueur des bras. I1 peut donc s'averer difficile de distinguer les autosomes les uns des autres par ces caracteres. La coloration des bandes C a revele la presence de petites bandes centromeriques chez les autosomes, alors que les chromosomes X et Y, beaucoup plus longs, possedaient des blocs extensifs et fonces de bandes C sur un fond intermediaire de coloration. La presque totalite de la fluorescence vive s'est manifestee chez les chromosomes du sexe, particulierement le chromosome X, dont le detail des bandes a ete remarquable. La resolution des bandes a ete fortement augmentee chez les cellules d'embryons en metaphase mitotique. La coloration a la quinacrine des chromosomes mitotiques a produit une fluorescence brillante dans les regions centromeriques des chromosomes 2, 3 et 4, ce qui a facilite leur identification. La luminosite de la fluorescence a ete nettement diminuee chez les chromosomes meiotiques, de meme que la resolution des bandes, mais elle a revele une asynapsis marquee des chromatides dans les regions de plus vive fluorescence des chromosomes du sexe. Les fluorochromes (quinacrine et DAPI), qui ont co!ore I'ADN riche en A . T, ont produit une coloration lumineuse dans une large proportion des chromosomes du sexe. A l'oppose, la chromomycine, qui se lie preferablement a I'ADN riche en G .C, a colore une proportion beaucoup plus faible des chromosomes du sexe que ce qui etait attendu d'une coloration reciproque. Ces donnees, ajoutees a celles de l'asynapsis, indiquent qu'une large proportion de l'heterochromatine des chromosomes du sexe possede des proprietes structurales inhabituelles. Mots clks : Chrysomya bezziana, mouche (( fil de vis n, caryotype, bandes C, fluorescence, heterochromatine. [Traduit par la redaction]
Introduction The Old World screwworm fly, Chrysomya bezziana, is a serious economic and health menace extending from South Africa through the Middle East and India to southeast Asia and New Guinea. A wide range of host animals and humans is attacked by this obligate parasite (Spradbery and Vanniasingham 1980). Although no populations have been established in Australia, its introduction presents a major economic threat to livestock industries. This, together with recent discoveries of live adults on ships originating from the Middle East and aircraft from India (Rajapaksa and Spradbery 1989), has provided impetus for development of a control strategy. Currently this is fulfilled by research into Printed in Canada / Imprime au Canada
the sterile insect release method (SIRM) (Knipling 1955) based in Port Moresby, Papua New Guinea. The New World screwworm, Cochliomyia homonivorax, is a similar economic and health problem currently the subject of a major SIRM control program in the United States and Mexico. Cochliomyia homonivorax has been the subject of speculation concerning genetic differentiation between natural populations (Lachance and Whitten 1986; Richardson et al. 1982). Such differentiation, if it existed in C. bezziana, would have great practical impact in the application of genetic control. In response to its introduction into Australia from different geographic regions, the cytogenetic study of C. bezziana has been intensified. A stan-
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632
GENOME, VOL. 34, 1991
dard polytene chromosome map has been compiled (D.G. Bedo, in preparation) and a comparison of banding patterns in populations throughout its range is in progress. Polytene chromosome analysis allows detailed study of fully replicated autosomal regions but fails to deal with underreplicated heterochromatin. Mitotic chromosomes must be used to study heterochromatin underreplicated in polytene cells. Measurement of chromosome lengths and arm ratios can show gross variation between populations, for example, the sex chromosomes of Co. homonivorax (LaChance and Whitten 1986). Finer resolution can be achieved by applying chromosome banding techniques to reveal cryptic variation within heterochromatin. Quinacrine staining and counterstain-enhanced fluorescence, which allow chromatin with a particular DNA content to be differentiated (Schweizer 1981, 1983), are particularly useful. For example, numerous bands revealed by fluorescence staining allow detailed cytogenetic analysis of the sex chromosomes of Drosophila melanogaster (Gatti and Pimpinelli 1983; Pimpinelli et al. 1985) and the Mediterranean fruit fly, Ceratitis capitata (Bedo 1989). Quinacrine banding applied to Co. homonivorax (McInnis 1981; McInnis et al. 1983; Richardson et al. 1982) detected sex chromosome variation in populations targeted for control by sterile insect release. Hybridization studies indicated that these differences represent polymorphism in natural populations (LaChance and Whitten 1986). This report details the characteristics of heterochromatic regions in mitotic and meiotic chromosomes of C. bezziana, using C-banding, quinacrine fluorescence, and counterstain-enhanced fluorescence. Materials and methods Flies Because the importation of live C. bezziana into Australia is prohibited, all mitotic and meiotic chromosome material was prepared during a visit to the New Guinea laboratory where mass-reared colonies are maintained. Chromosome preparation Air-dried mitotic chromosome preparations were made from third-instar larval brains as described by Bedo (1980). The same technique was modified to make air-dried preparations of whole embryos. Eggs were harvested approximately 3 h after laying and dechorionated by rolling them on the sticky side of a strip of adhesive tape. Dechorionated eggs were then placed in a drop of freshly mixed acetic alcohol and pierced with a sharp needle to allow penetration of the fixative. After 5 min the fixed eggs were transferred to a drop of 65% acetic acid on a slide and macerated with dissecting needles. The remaining steps are the same as those described earlier (Bedo 1980) for larval brain preparations. Chromosome measurements Chromosome arm measurements were made with vernier callipers from photographic prints of well-spread metaphase cells. The karyotype was visually ranked according to length before measurement. Chromosome arm ratios of all chromosomes were calculated and subjected to pairwise t-tests to compare centromere positions of all chromosomes. To quantify relative chromosome lengths, the percent of total haploid autosomal complement length (%TACL) for each autosome was calculated. Measurements of autosomes were also tested with paired t-tests for relative length differences. By only using the autosomes male and female cells could be pooled. The sex chromosomes were treated separately by calculating the length ratios of the X chromosome to the Y chromosome in male cells and the X chromosome to chromosome 2 in pooled male and female cells.
FIG. 1. Idiogram representing the karyotype of C. bezziana. The centromeres are indicated by constrictions. Nucleolar organiser regions in XL and YL are shown by gaps near the distal ends of the arms. Chromosome lengths and arm ratios are drawn in proportion.
Banding procedures C-banding and quinacrine staining were carried out as described by Bedo (1980). The counterstain-enhanced tristaining fluorescence method with chromomycin A! (CMA), 4,6-diamidino-2phenylindole (DAPI), and distamycin (Schweizer 1980, 1981, 1983) was also used. This allows CMA and distamycin-DAPI (DADAPI) staining to be observed in the same chromosome by simply changing excitation and barrier filter combinations. For sequential C-banding of tristained chromosomes, slides were destained in acetic ethanol for 24 h before applying C-banding procedures with reduced Ba(OH)2 pretreatment or staining in 2% Giemsa with no pretreatment (Bedo 1989).
Results Chrysomya bezziana has an XX/XY sex-chromosome system, designated here, using Drosophila conventions, as the first pair of the karyotype, and five pairs of autosomes numbered as pairs 2-6 (Figs. 1 and 2). Measurements (Table 1) show chromosomes 2 and 5 to be acrocentric, with chromosomes 3, 4, and 6 more metacentric in arm ratio. The autosomes form an evenly graded series in relative length, with chromosomes 3 and 4 being the most similar. Chromosome 3 is somewhat more acrocentric than chromosome 4, with a significantly different arm ratio (Tables 1 and 2). Chromosomes 3 and 4 are often difficult to distinguish visually, but as shown below, the application of quinacrine banding simplifies their identification. At over twice the length of the largest autosome pair, the X and Y chromosomes dominate the karyotype (Figs. 1 and 2; Table 3). A well-expressed secondary constriction occurs near the end of the Y chromosome long arm (YL) with an additional secondary constriction of variable expression more proximally in YL. The X chromosome has a small satellite at the end of the long arm (XL), which is easily seen only in extended prophase or early metaphase chromosomes. The prominent YL secondary constriction and the constriction separating the XL satellite are nucleolar organiser regions (NORs) containing ribosomal genes (D. G. Bedo, submitted).
TABLE1. Arm ratios ( + SD) and %TACL ( + SD) of 82 autosome complements from 58 mitotic metaphase cells and 9 second meiotic metaphase cells
TABLE2. Paired t-tests for differences between arm ratios and lengths of autosomes of the cells used in Table 1 Chromosome No. Chromosome No.
Chromosome No.
Arm ratio %TACL
1.83
1.43
( + 0.48)
(+ 0.03)
24.7
21.5
(+ 0.01)
(+ 0.02)
1.29 (k0.06) 20.2 ( + 0.03)
1.97
1.22
(k0.13)
(k0.05)
18.0
16.6
(k0.01)
(+ 0.01)
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TABLE3. Measurements (mean + SD) of X and Y chromosomes from cells used in Table 1 Arm ratio X*
Yt
X/Y ratio7
X/2 ratio*
*For these measurements averaged X chromosomes and second autosomes from 13 female cells were pooled with X chromosomes and averaged second autosome pairs from 45 male cells from Table 1. tForty-five X and Y chromosome pairs from the male mitotic metaphase cells were used.
The number of bands resolved in the sex chromosomes by all the techniques used varies greatly with the degree of chromosome contraction. Extended late prophase and early metaphase chromosomes are particularly suitable, showing more bands than contracted metaphase figures, in which banding groups tend to fuse into blocks. Embryo chromosomes are typically far less contracted than mitotic karyotypes and show the greatest resolution of bands.
C-banding Dark C-banding is produced mainly in the sex chromosomes (Figs. 2 and 10). Autosomal staining is limited to small centromeric bands, which do not consistently appear on every chromosome in a cell but are found on all autosomes in a sample of cells. This is probably a result of variations in the staining procedure sometimes resulting in overtreatment of these fine bands. The Y chromosome is darkly stained in the short arm (YS) and the proximal half of the long arm (YL). A region of reduced stain intensity occurs in the distal half of YL. This has intermediate "grey" staining, which is darker than the general pale staining of the autosomes. A distinct dark band obscures the secondary constriction region containing the Y chromosome NOR site near the end of the arm. An additional constriction, which is usually ill defined, lies close to the proximal end of the grey region. In the X chromosome, staining differentiation is more complex, with bands of varying intensity on a background of light- to medium-strength staining (Figs. 2 and 10). As in the Y chromosome, the distal half of XL has generally paler staining, with a dark band obscuring the NOR site in the secondary constriction at the tip of the arm. Faint bands may be distinguished in the distal grey region of XL, but these are not consistent. A large, intense C-band block marks the proximal end of the grey region. The centromere lies in an area containing four indistinct bands. The base of XS
2
3
4
5
6
NOTE:Arm ratios are compared above the diagonal; length comparisons are shown below the diagonal. *p
