The chromosomes of two Drosophila races: D. nasuta nasuta and D. nasuta albomicana

The microchromosomes of the totally cross fertile Drosophila races, D. nasuta nasuta and D. nasuta albomicana have been studied in nietaphase and polytene nuclei. In metaphase the microchromosome of D. n. albomicana is nearly five times longer than the homologous chromosome in D. n. nasuta. As shown by C-banding these length differences are mainly due to a massive addition of heterochromatin to the D. n. albomicana chromosome. In polytene nuclei these striking heterochromatin differences between the microchromosomes of the two Drosophila races cannot be observed. Analysis of the polytene banding pattern shows that the microchromosomes of both races differ by an inversion and by a duplication, present only in D. n, albomicana. The location and orientation of the duplicated regions in D. n. albomicana leads to a specific loop like chromosome configuration. On the basis of these differences within the Drosophila races studied it is assumed that the karyotype of D. n. albomicana is a more recent evolutionary product.     

Satellite DNA of Drosophila nasuta nasuta and D. n. albomicana: Localization in polytene and metaphase chromosomes

The DNA from the two Drosophila nasuta races, D. n. nasuta and D. n. albomicana was investigated by CsCl density gradient centrifugation. D. n. nasuta has one major AT-rich satellite DNA sequence with a density of 1.664g/cm³, while D. n. albomicana has at least three satellites with densities of 1.674g/cm³, 1.665g/cm³ and 1.661 g/cm³. The isolated satellite sequences hybridize in situ to all heterochromatic regions of all metaphase chromosomes of both races. In polytene chromosomes the satellite sequences hybridize exclusively to the chromocenter. All chromosomal regions hybridizing with the satellites show also bright quinacrine fluorescence.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. nasuta albomicans III. Localization of nucleolar organizer regions

In the two Drosophila races D. n. nasuta and D. n. albomicans the nucleolar-organizer regions (NORs) have been localized on metaphase chromosomes by a combined acid treatment and silver-staining technique. In both Drosophila races one NOR is present on the heterochromatic Y chromosomes and another on the microchromosomes. Such a NOR distribution has not yet been reported in Drosophila. It is suggested that this distribution has evolved from an original type with NORs on the X and Y chromosomes.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. nasuta albomicana

Interracial hybridization between Drosophila nasuta nasuta (2n=8) and D. n. albomicana (2n=6) has resulted in the evolution of two new karyotypic strains, called Cytoraces I and II. Males and females of Cytorace I have 2n=7 and 2n=6 respectively. The reconstituted karyotype is totally new in its composition, the chromosomes being drawn from both the parental races. The individuals of Cytorace II have 2n=6. Even though the chromosomes of the parental races are duly represented in the F₁, there is selective retention/elimination of certain chromosomes in the succeeding generations during which repatterning of the karyotype has taken place. Dynamics of each one of the parental chromosomes are presented and its implications re discussed.We dedicate this paper to the memory of the founder of our Department, the late Prof. M.R. Rajasekarasetty on the occasion of the Silver Jubilee of our Department     

The chromosomes of two Drosophila races: Drosophila nasuta nasuta and Drosophila nasuta albomicana V. Introgression and the evolution of new karyotypes

Reciprocal crosses were made between an Indian strain of D. n. nasuta (2n=8) and the Thailand strain of D. n. albomicana (2n=6). Hybrids were fertile. They were inbred for over four years. Later, the karyotypes of the hybrid populations were analysed. In the hybrid progeny of the cross between D. n. nasuta females and D. n. albomicana males, there were six types of kaotypes. Of these, only two types had a diploid content of chromosomes. They were males with 2n=7 and females with 2n= 8 , while others were aneuploids. This hybrid population is designated as Cytorace III. On the other hand, hybrid progeny of the reciprocal cross had 2n-8 in both males and females; and there was no karyotypic variation. This hybrid population is named as Cytorace IV. The composition of these new karyotypes of Cytorace III and IV have been presented and compared with those of Cytorace I and II reported by Ramachandra and Ranganath (1986).     

Estimation of inter-genotypic competitive ability of the parental races (Drosophila nasuta nasuta and D. n. albomicana) and of the newly evolved Cytoraces (I and II)

Interracial hybridization between D. n. nasuta (2 n = 8) and D. n.albomicana (2 n = 6) resulted in the formation of two new karyotypic strains denoted Cytorace I and Cytorace II. The karyotypes of each of these Cytoraces include chromosomal elements from both parental races (Ramachandra and Ranganath 1986a). The parental strains and the newly formed Cytoraces I and II were subjected to interspecific competition. The results reveal that all four experimental strains were competitively superior to the D. melanogaster tested strain. The study indicates certain degree of Cytogenetic divergence between parental and newly evolved genomes.     

Characterization of heterochromatin in Schistocerca gregaria by C- and N-banding methods

Mitotic and meiotic chromosomes of Schistocerca gregaria were C-, mild N- and strong N-banded. After C-banding, three out of eleven autosomes show, in addition to the centromeric C-bands, a second C-band. — The mild N-banding method produces a single N-band in each of only four chromosomes. With the exception of one N-band these mild N-bands correspond to the non-centromeric, second C-bands, indicating the heterochromatic character of at least three mild N-band regions. — The strong N-banding technique produces bands both at the C- and mild N-band positions and additionally a third band in one chromosome (M₈), not present after C- or mild N-banding. — The N-bands do not correspond to the nucleolus organizer regions. Because of the mechanisms of the N-banding methods, it is concluded that the centromeric heterochromatin, as well as the non-centromeric N-band regions, contain high quantities of non-histone proteins. Presumably a specific difference exists between the non-histone proteins in the centromeric and non-centromeric N-band regions because the centromeres are banded by the strong N-banding technique, but not after mild N-banding. It is concluded that the N-band regions (two exceptions) contain a heterochromatin type which has the following features in common with the -heterochromatin of Drosophila: C- as well as N-banding positive, high nonhistone protein content, repetitive and late replicating DNA. It is discussed whether the N-banded heterochromatin regions of Schistocerca contain that DNA fraction which is, like the Drosophila -heterochromatin, underreplicated in polyploid nuclei.     

A study of heterochromatin in Drosophila nasuta by the 5-bromodeoxyuridine-giemsa staining technique

Larval brain ganglia of Drosophila nasuta were cultured in vitro in the presence of 5-bromodeoxyuridine for 1 or 5 h at 24° C and the air-dried chromosome preparations stained by the Hoechst 33258-Giemsa technique to reveal bromodeoxyuridine induced sister chromatid differentiation. In 1 h as well as 5 h preparations, 10–15% of well spread metaphase plates show a sister chromatid differentiation in only C-band heterochromatin regions of different chromosomes. We infer that this sister chromatid differentiation in all heterochromatic regions is seen after bromodeoxyuridine incorporation for only one replication cycle and is related to the presence of asymmetric A-T rich satellite sequences in all the C-band regions of D. nasuta karyotype.     

Characterization of Drosophila heterochromatin

The C- and N-banding patterns of D. melanogaster, D. simulans, D. virilis, D. texana, D. ezoana and D. hydei were studied in comparison with quinacrine and Hoechst banding patterns. In all these Drosophila species the C bands correspond to the heterochromatin as revealed by the positive heteropycnosis in the prometaphase chromosomes. The N bands have the following characteristics: 1) they are always localized on the heterochromatin and generally do not correspond to the C bands; 2) they do not correspond to the nucleolar organizing regions; 3) they are inversely correlated with fluorescence, i.e., they correspond to regions which are scarcely, if at all, fluorescent after Hoechst 33258 or quinacrine staining; 4) they are localized both on regions containing AT rich satellite DNA and on those containing GC rich satellite DNA.     

N-banding in polytene chromosomes of Chironomus and Drosophila

The N-banding patterns of the polytene chromosomes of Drosophila melanogaster, Chironomus melanotus, Ch. th. thummi and Ch. th. thummi x Ch. th. piger were studied. In Chironomus the polytene N-banding patterns correspond to the polytene puffing patterns. This is revealed by comparison of the puffing and N-banding patterns of identical chromosomes. Size and staining intensity of the N-bands reflect the size of the puffs as shown by puff induction. There is no evidence that the N-bands are also located in Chironomus heterochromatin or are restricted to the nucleolar organizer regions. In Drosophila the -heterochromatin is strongly N-positive, whereas the -heterochromatin, as well as the Chironomus heterochromatin is not N-banded. Contrary to Chironomus, the puffs in Drosophila polytene chromosomes do not give rise selectively to well stained N-bands. — The N-banding method is interpreted to stain specifically non-histone protein which is (1) accumulated in genetically active chromosome regions and (2) present in a specific type of heterochromatin (-heterochromatin of Drosophila).     

Restriction enzyme digestion of heterochromatin inDrosophila nasuta

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.     

Heterochromatin diversity and cyclic responses to selective silver staining in Aedes aegypti (L.)

In interphase cells of Aedes aegypti (L.) (2n=4+ XX/XY), only the nucleolus responded to selective silver staining. The secondary constriction on chromosome 3 remained unresponsive at all times but the six centromeres were identified throughout mitosis from early prophase as well as those stages of meiosis subsequent to diplotene. The centromeric blocks were not synonymous with the pericentric heterochromatin revealed by C-banding. X chromosomes without an intercalary C-band were newly discovered in Ae. aegypti in the Bangalore strain. Sequential Q-or Hoechst 33258/C-banding in this and the Trinidad-30 strain revealed intercalary heterochromatin diversity within and between strains and also differences between intercalary and pericentric heterochromatin.     

Analysis of different banding patterns and late replicating regions in chromosomes of Allium cepa,A. sativum and A. nigrum

Different banding procedures and preferential Giemsa staining of late replicating DNA-rich regions were carried out in metaphase chromosomes of three species belonging to different sections of the genus Allium (A. cepa, A. sativum and A. nigrum). The banding, as well as the late replicating patterns were species-specific. The late replicating pattern proved to be, in all cases, the more detailed, and represented the highest percentage of the karyotype differentially stained. Lower percents of the karyotype positively stained were accounted for by C-banding, by modified C-banding and by N-banding. In A. cepa interphase nuclei the pattern of constitutive heterochromatin fitted well with that of late replicating DNA-rich regions, but the coincidence with that revealed by C-banding was only partial. This supports the suggestion that late replicating regions may be considered to be a special category of heterochromatin. On the other hand, it seems that not all C-banded material replicates at the end of the S phase. By the modified C-banding, stained centromere dots or small bands, as well as bands at the NORs are observed.     

Evolutionary experimentation through hybridization under laboratory condition in <Emphasis Type="Italic">Drosophila</Emphasis>: Evidence for Recombinational Speciation

Background  

Drosophila nasuta nasuta (2n = 8) and Drosophila nasuta albomicans (2n = 6) are a pair of sibling allopatric chromosomal cross-fertile races of the nasuta subgroup of immigrans species group of Drosophila. Interracial hybridization between these two races has given rise to new karyotypic strains called Cytorace 1 and Cytorace 2 (first phase). Further hybridization between Thailand strain of D. n. albomicans and D. n. nasuta of Coorg strain has resulted in the evolution of two more Cytoraces, namely Cytorace 3 and Cytorace 4 (second phase). The third phase Cytoraces (Cytorace 5 to Cytorace 16) have evolved through interracial hybridization among first, second phase Cytoraces along with parental races. Each of these Cytoraces is composed of recombined genomes of the parental races. Here, we have made an attempt to systematically assess the impact of hybridization on karyotypes, morphometric and life history traits in all 16 Cytoraces.     

Analysis of chromatin-associated fiber arrays

The distribution of constitutive heterochromatin has been investigated in four chromosomal races of the grasshopper Caledia captiva (2n= 23 /24 ) by the C-banding technique. Each of the four races was found to have a distinctive banding pattern which is associated with the inter-racial differences in chromosomal rearrangements. — The Ancestral race has a telocentric chromosome complement with large procentric C-bands which are structurally double on six pairs of chromosomes. The centromeres are unstained. — The General Purpose race has a C-banding pattern very similar to that seen in other Acridine grasshoppers with the majority of its chromosomes showing a centromeric localisation of the bands. — The two southern races, which show a complex polymorphism for presumed pericentric inversions on all twelve chromosomes, also show an unusually high level of interstitial and terminal C-bands. The different locations and numbers of these bands allow unambiguous identification of all the chromosome pairs within the complement. — In two cases, there is good evidence to indicate that a C-band redistribution between acrocentric and metacentric chromosomes has occurred by pericentric inversion. Furthermore, C-band variation on the long arm of the metacentric X-chromosome indicates the presence of a large paracentric inversion. This double inversion system has involved over 95% of the X-chromosome. — The interstitial and terminal C-bands probably have not resulted from heterochromatin movement within the complement but, more likely, have arisen by saltatory duplication of pre-existing sequences on the chromosome. — A new nomenclature system for banded chromosomes is proposed which allows most kinds of chromosomal restructuring and rearrangement to be adequately enumerated.     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

The period Gene: High Conservation of the Region Coding for Thr–Gly Dipeptides in the Drosophila nasuta Species Subgroup

In this study, the region corresponding to the Thr–Gly region of the period (per) gene in the Drosophila nasuta subgroup of species was sequenced. The results showed that this region was highly conserved in the D. nasuta subgroup. There were only nine variable sites found in this 300-bp-long region, all located in two small regions highly variable among Drosophila species. No length variation was observed either within this subgroup or in the Yunnan (YN) population of D. albomicans. The deduced amino acid sequences were identical for all 14 taxa in the D. nasuta subgroup, and a stretch of alternating Thr–Gly pairs was not observed in this subgroup. A phylogenetic tree was constructed. The clustering of some species was in general agreement with previous works, but it also raised some question on the phylogenetic relationship between the nasuta species. The data did not implicate the Thr–Gly region playing a role in behavioral isolation in this subgroup of Drosophila. Received: 8 February 1999 / Accepted: 22 April 1999     

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS–GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR ⁻ phenotype and induces specific swellings in heterochromatin.     

Decondensation of pericentric heterochromatin alters the sequence of centromere separation in mouse cells

The centromeres of a genome separate in a sequential, nonrandom manner that is apparently dependent upon the quantity and quality of pericentric heterochromatin. It is becoming increasingly clear that the biological properties of a centromere depend upon its physicochemical makeup, such as its tertiary structure, and not necessarily on its particular nucleotide sequence. To test this idea we altered the physical state of the AT-rich pericentric heterochromatin of mouse with Hoechst 33258 (bis-benzimidazole) and studied a biological parameter, viz., sequence of separation. We report that an alteration in the physical state of heterochromatin, i.e., decondensation, is accompanied by aberrations in the pattern of centromere separation. The most dramatic effect seems to be on chromosomes with large blocks of heterochromatin. Many chromosomes with large blocks of heterochromatin that, in untreated cells, separate late tend to separate early. Decondensation with Hoechst 33258 does not seem to alter the sequence of separation of inactive centromeres relative to that of active centromeres. These data indicate that alteration in the physical parameters of the pericentric heterochromatin might dispose the centromeres to errors. It is likely that this aberration results from early replication of the pericentric heterochromatin associated with active centromeres. Received: 24 August 1998; in revised form: 24 August 1998 / Accepted: 28 August 1998     

Rapid Evolution of a Few Members of Nasuta-Albomicans Complex of Drosophila: Study on Two Candidate Genes, Sod1 and Rpd3

Drosophila nasuta nasuta (2n = 8) and D. n. albomicans (2n = 6) are morphologically identical, cross fertile and karyotypically dissimilar pair of chromosomal races belonging to nasuta subgroup of immigrans group of Drosophila. Interracial hybridization between these two races yielded karyotypically stabilized newly evolved Cytoraces with new combinations of chromosomes and DNA content, and are called nasuta-albomicans complex of Drosophila. Along with many other features, striking plasticity in the lifespan has been observed in the karyotypically stabilized members of nasuta-albomicans complex of Drosophila. These findings provide a strong background to understand any changes at the molecular levels. In view of this, we cloned and characterized Sod1 and Rpd3 in the members of nasuta-albomicans complex of Drosophila. The evolution of Sod1 and Rpd3 in D. n. nasuta and D. n. albomicans is contrasting with the other species of Drosophila, at the level of synonymous mutations, intron variation, InDels and secondary structure changes in protein. In the members of NAC of Drosophila there were synonymous changes, variations in intron sequences of Sod1, whereas, in Rpd3, synonymous, nonsynonymous, intron variation, and secondary structure changes in protein were observed. The contrasting differences in the levels of Rpd3 (and Sir2) proteins were also noticed among short-lived and long-lived Cytoraces. The Cytoraces have exhibited not only specific changes in Sod1 and Rpd3, but also show pronounced changes in the levels of synthesis of these proteins, which indicates rapid evolution of these Cytoraces in laboratory. Further these Cytoraces have become a model system to understand the process of anagenesis.