Characteristics of Polytene Chromosomes from Ovarian Pseudonurse and Nurse Cells of otu Mutants

1.3.1. General Morphology, Chromosome Maps, Puffing Activity, and Protein Localization

The PNC and NC chromosomes from otu mutants vary greatly in their length, level of polyteny, and banding pattern (see Fig. 3). All the chromosomes can be classified by length into four major classes: "pompon" chromosomes (P) (see Fig. 3A,D) [for use of terms, see review by Zhimulev (26)], "half-pompons" (HP) (see Fig. 3B,C), "condensed" (C) (see Fig. 3G) and "normal" (N) (see Fig. 3E,F,H). P chromosomes are the shortest, HP are approximately twice as long as pompons (although still quite short), C chromosomes form a class that includes chromosomes with lengths between HP and N. Class N includes chromosomes with lengths close to those of salivary gland (SG) chromosomes. Also, several groups within each class were distinguished based on chromosome thickness (18).

The NCs of otu7/otu7 flies contain mainly nuclei with P and HP chromosomes. The otu11 homozygotes show all the types of chromosome morphology. A heteroallelic combination of otu7/otu11 results in a shift of the distribution in the direction of the normal class (see Table 1). Few chromosomes of P and HP classes occur, the percentage of condensed and normal chromosomes increases, and a new group of N extra-large chromosomes appears (see Fig. 3H) (18).

Initially, we worked with otu11 mutants, so photomaps were made using the N class chromosomes from NCs and PNCs of otu11/otu11 females (see Figs. 4-6). Thorough comparisons of the banding patterns of SG and PNC and NC chromosomes has revealed good correlations except for five regions, where no similarity occurs. These regions are shown by brackets on 2L at 24, 30, and 38 (see Fig. 5) and on 3L at 66, and on 3R at 95 (see Fig. 6). In many cases, the corresponding bands in SG chromosomes are split in PNC and NC chromosomes (21C1-2, 22C, 23D1-2, 23E1-2, 30AB, 48F, 51AB, 55C, 57E, 61C, 65D, 66E, 71A and 93F) (22,25,27).

The PNC and NC chromosomes develop a few large and many very small puffs. The main large puffs are 3CD, 7E, 8C, 11B, 22F, 42AB, 47A, 61AB, and 79D (18,22,25,27). Telomeric puff 61AB is the largest (see Fig. 7G). Chromosomes from the NCs of one egg chamber have the same puff pattern (see Fig. 7A-C), but chromosomes from different stages of egg chamber development show subtle differences in their puff patterns (see Fig. 7D-F) (Mal'ceva, unpublished data).

To investigate the location of induction of ecdysteron-induced puffs in polytene chromosomes from NCs, ovaries from females of otu7/otu11 genotype were incubated for 2-6 h at 25 °C or 3-24 h at 16°C in ecdysteron-containing organ culture medium. In another experiment, preincubation of NCs in an ecdysteron-free medium for 12 or 6 h was followed by incubation with ecdysteron for 8 or 6 h, respectively. NC chromosomes did not form puffs in response to ecdysone under these experimental conditions (28).

It is difficult to induce a heat-shock response in PNC and NC chromosomes. Flies homozygous for otu7 were temperature shocked to induce expression of the hsp genes, followed by in situ hybridization of the hsp26 probe to polytene chromosomes. In some PNC nuclei, a very faint signal was found (29). Long incubation in vitro of ovaries from otu7/otu11 flies in ecdysteron-containing medium causes formation of tiny hsp puffs in 63B, 67B, 83A, and 97D regions (28).

The PNC and NC chromosomes can be used for immunofluorescent localization of different proteins using antibodies. It is interesting to compare localization of the same proteins in somatic and germ-line cell polytene chromosomes. Data were obtained for Mod (product of the modulo gene) and HP1 [hetero-chromatin protein 1, product of the Su(var)2-5 gene]. In SG nuclei, anti-Mod antibodies prominently label the nucleolus, although pericentric heterochro-matin and the majority of euchromatic bands are also stained. In PNCs, there is

Giant Chromosomes With Label

Fig. 3. Types of chromosomal morphology of the 2L chromosome. In all cases (except c) the telomere is to the left. (A) middle-size pompon chromosome, (B) middle-size half-pompon chromosome, (C) large half-pompon chromosome, (D) extra-large pompon chromosome, (E) very thin, beaded polytene chromosome, (F) large normal chromosome, arrowheads show the weak points in 36C and D regions, (G) large-size condensed chromosome, arrowhead shows the weak point in 36D region, (H) extralarge chromosomes with normal banding pattern. Only three-fourths of the 2L chromosome is shown. The bar equals 10 |im. (From ref. 18. Copyright ©1997, used with permission, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.)

Fig. 3. Types of chromosomal morphology of the 2L chromosome. In all cases (except c) the telomere is to the left. (A) middle-size pompon chromosome, (B) middle-size half-pompon chromosome, (C) large half-pompon chromosome, (D) extra-large pompon chromosome, (E) very thin, beaded polytene chromosome, (F) large normal chromosome, arrowheads show the weak points in 36C and D regions, (G) large-size condensed chromosome, arrowhead shows the weak point in 36D region, (H) extralarge chromosomes with normal banding pattern. Only three-fourths of the 2L chromosome is shown. The bar equals 10 |im. (From ref. 18. Copyright ©1997, used with permission, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.)

intense staining of nucleoli, but the chromosome arms stain weakly, except for a few dense bands (30). Localization of HP1 also shows many differences between SG and PNC chromosomes in number and position of sites (Koryakov,

Fig. 4. Photomaps of the X chromosome (sections 1-20) (A,D) and chromosome 4 (sections 101-102) (B,C) from PNC of an otu11 mutant. The bracket marks granular material to the left of the 102A region, which often can be seen in chromosome 4. The bar equals 10 |im. (From ref. 27. Copyright ©1995, used with kind permission from Kluwer Academic Publishers.)

Fig. 4. Photomaps of the X chromosome (sections 1-20) (A,D) and chromosome 4 (sections 101-102) (B,C) from PNC of an otu11 mutant. The bracket marks granular material to the left of the 102A region, which often can be seen in chromosome 4. The bar equals 10 |im. (From ref. 27. Copyright ©1995, used with kind permission from Kluwer Academic Publishers.)

Polytene Heat Shock Puffs

Fig. 5. Photomaps of the left (sections 21-40) and right (sections 41-60) arms of chromosome 2 from PNC of an otu11 mutant. Brackets show regions where no similarity with the photomaps of SG polytene chromosomes was found. Arrowhead marks weak point in 39DE region. The bar equals 10 |im. (From ref. 27. Copyright ©1995, used with kind permission from Kluwer Academic Publishers.)

Fig. 5. Photomaps of the left (sections 21-40) and right (sections 41-60) arms of chromosome 2 from PNC of an otu11 mutant. Brackets show regions where no similarity with the photomaps of SG polytene chromosomes was found. Arrowhead marks weak point in 39DE region. The bar equals 10 |im. (From ref. 27. Copyright ©1995, used with kind permission from Kluwer Academic Publishers.)

Fig. 6. Photomaps of the left (sections 61-80) and right (sections 81-100) arms of chromosome 3 from PNC of an otu11 mutant. Brackets show regions where no similarity with the photomaps of SG polytene chromosomes was found. The bar equals 10 |im. (From ref. 27. Copyright ©1995, used with kind permission from Kluwer Academic Publishers.)

Fig. 6. Photomaps of the left (sections 61-80) and right (sections 81-100) arms of chromosome 3 from PNC of an otu11 mutant. Brackets show regions where no similarity with the photomaps of SG polytene chromosomes was found. The bar equals 10 |im. (From ref. 27. Copyright ©1995, used with kind permission from Kluwer Academic Publishers.)

Drosophila Polytene Chromosome Puff
Fig. 7. (A-C) Telomeric regions of the X chromosomes with different degrees of polyteny from one egg chamber have the same puff pattern. (D-F) Puff formation in 1A region. (G) Giant telomeric puff 61AB. The bar equals 10 |im. (Courtesy of N. I. Mal'ceva, unpublished data.)

unpublished data). An example of HP1 localization on a PNC polytene chromosome can be seen in Fig. 8.

1.3.2. Heterochromatin Morphology in Pseudonurse and Nurse Cell Chromosomes

Pericentric regions of SG polytene chromosomes are usually involved in nonhomologous associations that result in a structure called the chromocenter. In PNC and NC polytene chromosomes, the chromocenter consists of blocks of polytenized material connected by tiny threads, which can be easily broken during squashing (see ref. 20, Fig. 8B). Sometimes, bands and even puffs can be seen inside these blocks (see Fig. 9) (27,31-33). The pericentric region of the X chromosome in PNC and NC contains a characteristic banding pattern in region 20A-F (see Fig. 4D), which is rarely seen in SG chromosomes (27,31). Using a number of chromosome rearrangements and DNA clones with known localization in mitotic heterochromatin, a correspondence was found between polytenized material in PNC autosomes and differentially stained blocks in mitotic chromosomes. The polytenized material in pericentric regions of chromosomes 2 and 3 originates from proximal mitotic heterochromatin rather than proximal euchromatin or material from eu-heterochromatic junction regions (32-34, Domanitskaya and Koryakov, unpublished data).

The genetic inactivation of euchromatic genes placed next to pericentric hetero-chromatin by a chromosomal rearrangement is accompanied by the compaction of corresponding euchromatic chromosome regions (so-called position-effect variegation). A comparative study of the manifestation of position-effect variegation for the polytene chromosomes of SGs and NCs was made using the Dp(1;1)pn2b and Dp(1;f)1337 rearrangements. The percentage frequencies of block formation in the SG and NC nuclei for Dp(1;1)pn2b were 92.6% vs 15.8%, respectively; for Dp(1;f)1337, these values were 56.8% vs 9.7%, respectively. Therefore, pericentric heterochromatin belonging to germ-line chromosomes is in a configuration that is far less likely to inactivate inserted segments of euchromatin than is heterochromatin from somatic chromosomes (18).

In polytene chromosomes, there are several sites, which, because of their characteristics, are believed to be the sites of intercalary heterochromatin (35). Usually, they form weak points where chromosome breaks occur, and they tend to form ectopic contacts with nonhomologous sites and pericentric regions of chromosomes. In PNC chromosomes, breaks were observed in at least 11 sites of the X chromosome, 8 sites of chromosome 2L, 5 sites of 2R, 5 sites of 3L, and 13 sites of 3R, but their frequency is substantially lower in PNC chromosomes than in those from SG cells. In regions 36C, 36D, 39E, and 56F, frequencies of breaks are comparable with those in SG chromosomes (see Figs. 3F,G and 5) (27; Koryakov, unpublished data).

A

1dfe*

' X*

I

•4

1» _

A

M

* ~ r * t

t

a

A \2

ft

Fig. 8. The distal part of the X chromosome from an otu11 homozygote with a fluorescence microscope. (A) The chromosome was stained with DAPI, so the bright blocks and bands localize DNA. (B) The chromosome was stained with the CIA9 antibody against HP1. A comparison of A and B localizes brightest signals of HP1 to regions 1A, 1F, 2D, 3A, 3C, 3E, and 3F. (Courtesy of D. E. Koryakov, unpublished data.) (See color plate 4 in the insert following p. 242.)

Fig. 8. The distal part of the X chromosome from an otu11 homozygote with a fluorescence microscope. (A) The chromosome was stained with DAPI, so the bright blocks and bands localize DNA. (B) The chromosome was stained with the CIA9 antibody against HP1. A comparison of A and B localizes brightest signals of HP1 to regions 1A, 1F, 2D, 3A, 3C, 3E, and 3F. (Courtesy of D. E. Koryakov, unpublished data.) (See color plate 4 in the insert following p. 242.)

The frequency of formation of ectopic contacts between different PNC chromosome regions is 10 times less than in the polytene chromosomes of SG cells. The regions that form ectopic contacts in PNC chromosomes are 2C, 3AB, 9A, 10A, 11A, 11D, 12D, 12E, 26C, 28D, 48D, 49A, 58A, 58F, 59A, 60E, 66E, 67CD, 67E, 69D, 70A, 75F, 76B, 98D, and 99A (27).

In PNC chromosomes from both otu and fs(2)B mutants, asynapsis occurs both between the two homologs and between the bundles of chromatids within each homolog. One of the main features offs(2)B chromosomes is the incomplete association (or secondary splitting) of the chromatids. Sometimes, complex reconjugations of parts of different homologs are seen (see Fig. 10). Such

Fig. 9. Structure of the pericentric heterochromatin of chromosome 3. (A) Part of the chromocenter with pericentric regions of SG chromosome 3 from an otu11 homozygote; (B) PNC chromosome 3 from an otu11 homozygote carrying Dp(1;f)1337. The brackets mark heterochromatic material. The bar equals 10 |im. (Courtesy of D.E. Koryakov and N.I. Mal'ceva, unpublished data.)

Fig. 9. Structure of the pericentric heterochromatin of chromosome 3. (A) Part of the chromocenter with pericentric regions of SG chromosome 3 from an otu11 homozygote; (B) PNC chromosome 3 from an otu11 homozygote carrying Dp(1;f)1337. The brackets mark heterochromatic material. The bar equals 10 |im. (Courtesy of D.E. Koryakov and N.I. Mal'ceva, unpublished data.)

splitting of individual homologs is very rare in NC chromosomes of otu mutants, with the exception of otu11/otu14 (Mal'ceva, unpublished data). Asynapsis between homologs in PNC chromosomes in all of these mutants is much more frequent than in SG chromosomes, especially in regions 19-20, 39-40, 41-42, 79-80, and 81 (27).

From the above, it is clear that polytene chromosomes of otu mutants can serve as a useful model for studying features of gene expression and eu- and heterochromatin behavior.

Was this article helpful?

0 0

Post a comment