Cytogenetic Mapping of Enzyme Loci on Chromosomes J and U of Drosophila Subobscura

CYTOGENETIC MAPPING OF ENZYME LOCI ON CHROMOSOMES J AND U OF DROSOPHILA SUBOBSCURA

W. PINSKER AND D. SPERLICH Lehrstuhl fur Populationsgenetik, Institut fur Biologie 11, Universitat Tubingen, Auf der Morgenstelle 28, 0-7400Tubingen, Federal Republic of Germany Manuscript received April 24, 1984 Revised copy accepted July 20, 1984

ABSTRACT Enzyme loci located on chromosome J and U were mapped cytologically by means of a Y translocation technique. A linkage map of the two chromosomes was established in a parallel experiment and the recombination frequency in different regions of the chromosomes determined. A comparison of the cytogenetic localization of the enzyme genes in D. subobscura and D. melanogaster indicates that many paracentric inversions must have taken place in the course of divergent evolution. However, no displacements of genes from one element to another due to pericentric inversions, reciprocal translocations or transposing elements can be observed. In spite of the large number of structural rearrangements that have occurred in the phylogeny of the genus Drosophila, gross similarities of banding pattern in homologous regions of the chromosomes of the two species become apparent.

ITH the introduction of gel electrophoresis, enzyme loci have become W very important marker genes for population genetic studies. Allozyme variation has proved especially useful for the estimation of the amount of genetic variation and for the calculation of genetic relationship between populations, species and other taxa. In addition, a number of studies have been devoted to the search for nonrandom associations between alleles of different loci or linkage disequilibrium, maintained by selective forces favoring certain allele associations by epistatic interaction. In spite of the large effort invested, unambiguous evidence for stable linkage disequilibria due to gene interaction is still scarce. In Drosophila subobscura the only good example has been re- ported by ZOUROS and KRIMBAS(1973) for the loci Ao and Xdh. On the other hand, nonrandom associations between genes and chromosomal inversions appear rather common. In the inversion polymorphic species D. subobscura, gene inversion associations have been described for the chromosomes A (CABRERAet al. 1983), E (LOUKASand KRIMBAS 1975), J (PREVOSTI et al. 1982), 0 (CHARLESWORTHet al. 1979) and U (PINSKERand SPERLICH 1982). It can be assumed that practically all of the genes involved in these associations are located either inside the respective inversions or at least close to one or other of their breakpoints. Thus, inversions represent not only mere structural changes in the sequence of the genes but genetically differentiated

Genetics 108: 913-926 December, 1984. 914 W. PINSKER AND D. SPERLICH regions of the chromosomes. Since gene flow between the inverted regions of different gene arrangements is assumed to be minimal because of the virtual absence of recombination in heterokaryotypes, gene arrangements can be considered to have their own isolated gene pools. As a consequence, differences with respect to allelic frequencies for the loci within the inversions can be found (PINSKERand SPERLICH1981). Two mechanisms have been proposed to explain the cause of this genetic differentiation occurring between chromosomal structures: The historical explanation advanced by ISHII and CHARLES- WORTH (1977) is the simplest and assumes only a chromosomal founder effect brought about by the formation of a new inversion. The second hypothesis, which is mainly due to Lewontin and his group (PRAKASHand LEWONTIN 1968), assumes coadaptation producing heterotic effects in heterokaryotypes due to the effects of the relationally balanced chromosome segments. Evidence in favor of both theories exists, and in nature probably both mechanisms contribute to the diversification of gene arrangements. However, for a careful and reliable investigation of the problem it is necessary to know the exact cytological positions of the gene loci under study, not only relatively with respect to other genes on the same chromosome but also cytologically with respect to the centromeres and the inversion breakpoints. In this study we have cytologically localized 11 genes of D. subobscura. Al- though D. subobscura is widely used for population genetic sudies, the existing linkage maps are still rudimentary or even partly incorrect. Cytogenetic information on the location of the enzyme loci is only available in form of linkage data with inversions. Using a recently developed Y autosome translocation technique (SPERLICHand PINSKER1984), we have been able to map the enzyme loci on the giant chromosomes more accurately. In this paper we describe the mapping of the autosomes J and U for which the available information is especially poor. A linkage map for chromosome J has been published previ- ously (LOUKASet al. 1979), but the recombination data given there are incomplete and the assumed sequence of genes is ambiguous. For chromosome U no linkage map has hitherto been available. Consequently, recombinational linkage maps for these two chromosomes were also constructed to supplement the results of the cytogenetic study. Furthermore, the accurate cytological localization of the enzyme loci allows us to investigate chromosomal homologies at the interspecific level. In species that cannot be intercrossed, chromosomal homologies can be only found by searching for similarities in the banding pattern of the giant chromosomes or, more precisely, with the aid of homologous marker genes. The first approach is somewhat subjective and useful only for groups of species that are closely related and differ in chromosome structure only by a few rearrangements. For the second approach, well-defined structural genes of orthologous relationship are a prerequisite. This requirement is perfectly fulfilled by the enzyme loci. Since most of the enzyme loci have been already localized cytologically in D. melanogaster by means of deletion mapping, a knowledge of their cytogenetic position in D. subobscura allows a direct coordination of chromosomal segments of the two species and, consequently, the chromosomal evolution in the two species can be inferred. CYTOGENETIC MAPPING IN DROSOPHILA 915

MATERIALS AND METHODS Strains: All allozyme strains used in the following experiments are derived from natural population samples collected between 1975 and 1983 at different localities from Europe and North Africa. Homozygous strains for rare allozyme variants were established for the following enzymes: alcohol dehydrogenase (Adh), alkaline phosphatase (APh), diaphorase (Diu), esterase-3 (Est-3), a- glycerophosphate dehydrogenase (aGpdh), isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh) and phosphoglucomutase (Pgm).In addition three morphological mutants were provided by A. PREVOSTI(Barcelona, Spain): the eye pigment mutants cinnabar (cn) and maroon (ma), both located on chromosomej, and the wing vein mutant net (net), which is on chromosome U. For the translocation experiments the following marker chromosomes were constructed: ChromosomeJ: (a) Aph"', IdhIo5, Pgm'', +" (b) Aph'", Idh"', Pgm105,cn Chromosome V (a) aGpdhlo4, Mdhn6, (b) aGpdh'", MdhQ6,net Translocation technique: This method, originally developed by TAN(1 937), has been recently adapted for the localization of enzyme loci by SPERLICHand PINSKER(1 984). Translocations between the Y chromosome and an autosome are induced by X-rays, and irradiated males of the wild-type strains (a) are crossed to females homozygous for a recessive mutant (b). F1 males are then individually backcrossed to mutant females (b). The occurrence of a translocation can be detected by phenotypic examination of the progeny since the autosomal genes transferred to the Y chromosome display patroclinous inheritance. The translocated enzyme genes can be determined by electrophoretic investigation of the F2 or of the offspring of further crosses. A cytological analysis of the polytene chromosomes reveals the size and breakpoints of the translocated segment. Electrophoretic techniques: Electrophoresis was carried out in horizontal starch gels according to the methods of AYALAet al. (1972) and LOUKASand KRIMBAS(1980). The designation of allozymes follows the system proposed by SAURAet al. (1973). In zymograms of the Aph allozymes the staining intensity of the bands was measured with a Quick Scan Densitometer (Desaga, Heidelberg, Federal Republic of Germany). For this purpose the starch gels were made transparent in 5% glycerol using the method of NUMACHI(1 98 1). Cytological analysis: For the determination of the translocation breakpoints the polytene salivary gland chromosomes of male larvae were stained with acetic orcein. The subsections of the D. subobscura chromosomes are designated according to the chromosome map of KUNZE-MUHL and MULLER(1 958). Mitotic metaphase chromosomes were prepared from larval neuroblast cells using the squash technique proposed by GUESTand Hsu (1973).

RESULTS Photographic maps of the polytene chromosomes U and J of D. subobscura are presented in Figure 1, which shows the positions of the translocation breakpoints, the breakpoints of some common inversions and the location of the enzyme genes. Each gene locus is mapped between the adjacent translocation breakpoints. An example of the mode of cytological localization is given in Figure 2 which describes the situation found with translocation J7, where the distal end of chromosome J has been translocated to the Y chromosome. In the offspring of males carrying this translocation two types of males occur: balanced genotypes and aneuploids. When the appropriate marker strains for the backcrosses are used, the genotype of the aneuploids reveals whether a gene has been translocated to the Y chromosome or not. In the case of translocation J7 the irradiated J chromosome carried the enzyme variants Aph"', ZdhIo5 and Pgm". The aneuploid males obtained after two backcrosses to the marker strains Aph"', Zdh'", PgmIo5 and Aph'", Zdh'05, Pgm'", respectively, have three copies of the Pgm gene (97/101/105), three copies of the Idh gene (101/105/105) but only two copies of the Aph locus (100/100) according to Chr. U Chr: J

TL -18

c EST-3 TL AOH 9 otGPOH -11 8 c 30 E.

5 20

0 -17 -C t- APH -.22 . -16 -- c 6- OIA * A4 3b - 7 10 ----a- IOH ....-.- MOH 21 -13 12 s.2s

net

T FIGURE1 .-Photographic maps of the polytene chromosomes U andJ of D. subobsrura showing the position of eight eniyme loci and three genes for visible characters. The region containing the gene is defined by the adjacent translocation breakpoints (TL). The breakpoints of the conimon inversions UI, U2, U8 and JI are also indicated (C = centromere, T = telomere). 916 CYTOGENETIC MAPPING IN DROSOPHILA 91 7 allozyme chromosome I genotypes pattern

aneuploids

PGM 100 I00 APH IPH 105 101 I05 IDH 97 105 101 Fa4

APH

balanced

t, A BA B B

xx) APH

105 IDH

101 F'GM I 97 FIGURE2.-Chromosomal and allozyme patterns of aneuploid and balanced genotypes of males carrying Y autosome translocations. The distal end of chromosome J is translocated to the Y chromosome. The aneuploid males are trisomic for this section as can be easily recognized from the photograph of the polytene chromosome. The genes located in the translocated part of chro- mosonieJ can be determined by comparing the allozyme pattern of the aneuploid (A) and the balanced genotypes (B) in the zymogram. the allozyme pattern depicted in Figure 2. Thus, the breakpoint of translocation J7 separates the Aph locus from the two other genes which must be located in the distal part of autosome J. For the successful application of this method two requirements must be met: expression of the translocated genes and survival of the aneuploids. Although it is hypothetically possible that the translocation of a gene to the heterochro- matic Y chromosome may result in its inactivation, the allozymes coded by the translocated loci were always detected in the electrophoretic analysis. However, as a further confirmation, a more detailed investigation of this problem was carried out with the Aph locus. Heterozygotes for the alleles 100 and 111 were constructed and run side by side in the same gel with individuals of the balanced genotype, having the allele l l l translocated to the Y chromosome and the allele 100 on the autosome. After electrophoresis and staining for Aph activity all individuals displayed the three-banded allozyme pattern typical for a dimeric enzyme, consisting of the two homodimers 100/100 and 11 1/111 and the heterodimer 100/111. The staining intensity of the bands was evalu- ated densitometrically for each individual, and the ratio between the 11 1/111 and the 100/100 homodimers was determined. In both the normal heterozygotes and the heterozygotes with the Y translocation a higher absorption was measured with the 1 1 1 /I11 homodimer band, with a ratio of 1.47 -C 0.24 and 1.53 f 0.22, respectively. No difference, however, could be detected between the two genotypes. The relative activity of the Aph"' allele is apparently the 918 W. PINSKER AND D. SPERLICH same, irrespective of whether the gene is on the Y chromosome or on the autosome. A more severe problem arises from the reduced viability of the aneuploids observed in about 25% of the translocation strains. If the frequency of the aneuploids is less than 10% of the expected 1:l ratio, the electrophoretic analysis becomes too laborious. In some cases the aneuploids do not survive the larval stage, but this is not so important since most of the enzymes can be analyzed in larvae too. The size of the translocated segment apparently does not affect the viability of the aneuploids. The breakpoints of the translocations J18 and U9 are close to the centromeres which means that almost the whole autosomes have been translocated. Nevertheless, in both cases viable aneuploid larvae and adults are found. Although the Y chromosome itself is not visible in the preparations of the polytene chromosomes, the Y autosome translocations are thought to be reciprocal. The distal end of the autosome becomes attached to the deleted Y chromosome and vice versa. Mitotic metaphase preparations of males carrying a translocation support this interpretation (Figure 3). As a consequence, males with balanced genotypes possess the genetic information of a complete Y chromosome and, therefore, are fully fertile. The aneuploids, on the other hand, lack the distal part of the Y chromosome (Figure 2) and, thus, are sometimes found to be sterile due to the loss of fertility genes. Since the aneuploids serve only as material for the electrophoretic analysis and are not involved in the crossing procedure, their fertility is irrelevant. Among the 40 translocations produced for this study all but one were translocations of the distal end of the autosomes to the Y chromosome. The only exception was found with translocation U3 where the central section of chromosome U was inserted into the Y chromosome (breakpoints 3a and 3b in Figure 1). In general the meiotic behavior of X and Y chromosomes was normal in spite of the translocations. One remarkable exception occurred with translocation U20, where two different types of aneuploid males appeared: (1) aneuploids for the distal end of the chromosome U (which were trisomic for Mdh as shown from a triallelic cross) and (2) aneuploids for the proximal part (trisomic for aGpdh). Since the aneuploid males of the latter genotype were fertile, it has to be assumed that most of the Y chromosome became attached to the proximal region of chromosome U. The occurrence of two different types of aneuploids poses the question how the chromosomes segregate during meiosis. One explanation could be that in this special case a trivalent is formed by the intact autosome U and the two reciprocal translocation chromosomes, whereas the X chromosome functions as an univalent. The chromosomes of the trivalent can disjoin in three different ways giving rise to 12 different types of gametes, provided that the X chromosome is distributed independently. Four of these gametic types will have incomplete haploid genomes causing lethality of the zygotes. The rest will result in viable zygotes of eight different genotypes, among them chromosomal combinations like XO males, XXY females and aneuploid females. Some of these unusual genotypes were discovered in the electrophoretic analysis of the offspring of this translocation strain. CYTOGENETIC MAPPING IN DROSOPHILA 91 9 P

FIGURE3.-Mitotic metaphase chromosomes of D. subobscuru males. a, Normal male; the Y chrornosonie is easily recognized by its unseparated chromatids. b, Male heterozygous for a recip rocal U-Y translocatioii. Almost the entire U chromosome is translocated to the centromere of the Y chroniosorne (Y-U). The reciprocal U-Y chromosome consisting of the U centromere and a large part of the Y chromosme appears as an untouched Y chromosome with unseparated chromatids. c. Male heterozygous for a reciprocal 0-Y translocation. A large part of the Y is attached to almost the entire chromosome 0. The ring configuration is produced by the separated chromatids of chromosome 0 brought into contact at the distal end by the Y chromatids. The small Y-0 chre mosome consists of the Y centromere and the 0 telomere.

An x-ray dose of 9000 rads delivered over a period of 30 sec was used to induce the translocations, the highest dose tolerated by D. subobscuru without severe effects on the survival and fertility of the irradiated males. For chromosome J, 19 different Y-J translocations were obtained from 1260 single male crosses, a yield of 1.4%. For chromosome U the number of Y-U translocations was 21 among 840 single male cultures tested (2.5%). The difference in the percentage of the translocations found for the two autosomes is not statistically significant, although chromosome-specific tolerance of breakage might exist. Preliminary experiments show that Y translocations can be produced with the two other autosomes of D. subobscura too and that viable aneuploids can be obtained. Although the sequence of the enzyme genes on the two chromosomes can be derived from the translocation experiments, the genes were also mapped recombinationally in order to verify the cytological data. It was not the aim of this investigation to get highly accurate map distances but only to establish the order of the genes along the chromosome unambiguously. Thus, overlapping three-point testcrosses were carried out for all loci included in our map. The results are summarized in Tables 1 and 2. With chromosome U the inversion polymorphism of this chromosome was a major obstacle. Since the three bio- chemical markers Adh, dpdhand Mdh do not display much allozyme variation in natural populations (PINSKERand SPERLICH1979), we were not able to establish all of the marker strains necessary to obtain recombination values for the different gene arrangements separately. Hence, the position of the genes with respect to the inversions cannot be decided unequivocally from the map ping data. The map presented in Figure 4 can be constructed only with the 920 W. PINSKER AND D. SPERLICH

TABLE 1

Recombination values between the marker genes used on chromosome U of D. subobscura in heterozygote females of dayerent karyotypes

Karyotype in Corrected chromosome No. of Recombinants map U Loci individuals (% distance StlSt Adh-CiGpdh 263 0.8 f 0.5 0.8 StlSt Adh-Mdh 263 49.4 f 3.1 31% CiGpdh-Mdh 263 48.6 f 3.1 st/r + 2 aGpdh-Mdh 88 0 0.0 Stll + 2 Mdh-net 88 0 0.0 St/l + 2 aGpdh-net 88 0 0.0 St/l + 2 + 8 aGfdh-Mdh 386 0.3 f 0.3 0.3 I + 211 + 2 + 8 Mdh-net 96 47.9 f 5.1 The map distances are corrected for multiple crossing over according to SUZUKI,GRIFFITHS and LEWONTIN(1981). For map distances based on recombination values greater than 40% a correction is considered unreliable.

TABLE 2

Recombination values between the marker genes used on chromosome J of D. subobscura

Karyotype in chromosome No. of Corrected J Markers individuals Recombinants (W) map distance Stll Est-jr-J, 10.9” 12.3 Stll JI-APh I30 28.5 f 4.0 42.2 Stl I ma-Aph 200 10.0 f 2.1 11.2 Stll ma-ldh 258 34.5 f 3.0 Stll Aph-Idh 174 21.3 f 3.1 Stll Aph-Dia 174 8.0 f 2.1 8.7 Stll Dia-Idh 174 14.4 f 2.7 17.0 Stll Dia-Pgm 174 40.2 f 3.7 Stll Idh-cn 414 36.3 f 2.4 64.4 Stll Idh-PP 414 37.4 f 2.4 Stll cn-Pgm 414 3.2 f 0.9 3.2 The female parents were heterozygous for the gene arrangements Js,/JI in all crosses. Map distances were corrected only for adjacent loci. Recombination value published by LOUKASet al. (1979).

additional knowledge from the translocation experiments. With chromosome J the situation was less complicated since all of the loci under study were situated outside of the common inversion J,. Nevertheless, our results show that recombination between two loci can be influenced by the structural heterozygosity on the other chromosomes as well (SCHULZand REDFIELD1951). An example is given in Table 3. Females heterozygous for complex inversions on the autosomes E, 0 and U proved to have a higher crossing over frequency on chromosome J than structurally homozygous females. Thus, the genetic back- ground has to be taken into consideration for the correct interpretation of linkage data, at least in a species with such pronounced chromosomal polymorphism as D. subobscura. CYTOGENETIC MAPPING IN DROSOPHILA 921

0 sub D nrl 0-sub D me1

U 2L J 3L

P - ADH -EST-3 APH ‘~GPDH - ADH - st PGM I1 - EST-6

- MOH

-ma - -m IDH se -DIA

- IDH - ctGPM “2

/NOH

‘net cn

-net -FliM FIGURE4.-Comparison of the genetic maps of D. subobscura and D. melanoguster. The map of D. subobscura is based on the recombination values given in Tables I and 2. For the comparison of the sequence of the loci the chromosomes are depicted in equal size. Measured in map units, however, the length of the D. subobscura chromosomes would be twice the length of the chromosome arms of D. melanogaster due to a considerably higher recombination rate.

TABLE 3 Recombination values beheen the genetic markers on chromosome J in females with dafferent genetic backgrounds

% recombinants Chromosomal No. of heterozygosity J1-W Aph-Idh Idh-Pgm individuals

~~ 0 19.0 f 5.4 9.4 f 4.0 22.6 f 5.7 53 1 33.2 * 3.5 15.8 f 2.7 27.7 f 3.3 184 2 28.6 k 5.4 20.0 f 4.8 32.9 ? 5.6 70 0 = homozygous for the standard gene arrangements on chromosome E, 0 and U; 1 = heterozygous for standard and one of the gene arrangements E1+2+9,03+4+8 or U1+2+8;2 = heterozygous for standard and two of these gene arrangements. The difference between class 0 and the pooled data of the classes 1 and 2 is significant (x2 = 5.72, d.f. = 1, P < 0.05).

DISCUSSION These data clearly demonstrate that cytogenetic mapping is a good way to obtain reliable information on the position of genes on the chromosomes. Linkage data alone are often misleading, especially if inversions are involved. The Mdh locus may serve as an example: From genetic linkage data (SPERLICH, PINSKERand EL-ABIDINSALAM 1976) and from nonrandom associations be- 922 W. PINSKER AND D. SPERLICH tween inversions and Mdh alleles observed in nature (PINSKERand SPERLICH 1982), there appeared to be no doubt that the gene coding for Mdh must be located inside of the inversion US. This assumption, however, is refuted by the cytological results, which show that the gene must be situated near the distal end of chromosome U between translocation breakpoints U10 and U21 (Figure 1). The different recombination values between Mdh and net obtained in different karyotypes (Table 1) indicate that Mdh must be located somewhere inside the complex inversion U1+2. The close linkage between the inversion U8 and Mdh can be explained only by the fact that inversion Us is superimposed to the two inversions U1 and U,. If one converts the standard gene arrangement into the gene arrangement U1+2+8 (the inversion is found only in this com- bination), the Mdh locus is brought into the neighborhood of U8 due to the reversal of the sequence of the genes in the U1 region. Another source of misinterpretation of the cytological position of genes results from the fact that recombination frequency is not constant for all regions of a chromosome. According to the linkage data aGpdh appears to be closely linked to the inversion complex U1+2. On the cytogenetic map, however, the position is near the centromere. Thus, recombination must be greatly sup- pressed in this section of the chromosome. A similar phenomenon can be observed in D. melanogaster when the linkage map is compared with the cytogenetic map (LINDSLEYand GRELL1968). Here too, recombination is reduced in the proximal parts of the chromosome arms. As we have pointed out, enzyme loci are excellent markers for inferring chromosomal evolution. In Figure 4 the genetic maps of the chromosome U andJ of D. subobscura are compared with the maps of the homologous elements of D. melanogaster (LINDSLEYand GRELL 1968; O’BRIEN and MACINTYRE 1978): chromosome U corresponds with the left arm of chromosome 2 of D. melanogaster and chromosome J with the left arm of chromosome 3. The homology between the genes of the two species seems to be undisputed with the enzyme loci Adh, aGpdh, Mdh, Aph, Idh and Pgm. The homology between Est-3 of D. subobscura and Est-6 of D. melanogaster requires further confirmation, although both subunit structure (both enzymes are monomeric) and sub- strate specificity (P-esterases) indicate homology. A gene homologous to the diaphorase (Dia) locus of D. subobscura first described by LOUKASet al. (1979) has not yet been mapped in D. melanogaster. With the visible markers, cn of D. subobscura seems to correspond with st in D. melanogaster (both mutants lack the brown xanthommatin eye pigment component and both are closely linked to the Pgm locus). The homology between ma of D. subobscura and se of D. melanogaster appears also reasonable since both mutants possess the additional eye pigment sepiapterin in great quantity and lack the drosopterins. With the wing vein mutant net of both species the supposed homology is derived from the phenotypic similarity only, although the location of the gene on the distal ends of the respective chromosomes also suggests homology. When the order of the homologous genes on the respective chromosomal elements of the two species is compared, it becomes evident that a high number of paracentric inversions must have been fixed in the two diverging phyloge- netic branches of the two species. When the pronounced inversion polymor- CYTOGENETIC MAPPING IN DROSOPHILA 923 phism found in recent populations of both species is taken into account, the amount of chromosomal differentiation is not surprising. On the other hand, it seems remarkable that the cytological position of the genes studied does not indicate that exchange between different chromosomal elements has ever taken place, either by means of reciprocal autosome-autosome translocations or by fixed pericentric inversions in the two large metacentric autosomes of the D. melanogaster phylad where two primordially separated elements have been com- bined by centric fusion. Even in the absence of chromosomal rearrangements, some movement in the position of loci may be expected from association with transposable elements (GEHRINGand PARO 1980). Yet, our observations, although restricted to a few loci, do not provide evidence that this sort of gene movement occurs very frequently in evolution. This is in accordance with the conclusions of STURTEVANTand TAN(1937) and of STURTEVANTand NOVITSKI (1 94 1) who compared the position of homologous genes determining visible characters in D. pseudoobscura and D. melanogaster. Recent studies, using ra- dioactive single copy DNA for in situ hybridization on the polytene chromosomes of several species of the D. obscura group, also support the assumption. Tandem repetitive genes, however, seem to behave differently (STEINEMANN, PINSKERand SPERLICH1984). In view of the many fixed inversions separating the chromosomal elements of D. melanogaster and D. subobscura it appears a futile enterprise to search for homologies in the banding patterns of the polytene chromosomes. The size of eventually preserved regions of the ancestral chromosomes is supposed to be rather small and the directional alignment in the two species could be different. Yet, the situation is not so complicated as expected. Once the cytogenetic location of orthologous genes is known, they can be used as starting points for the comparison of the banding pattern. In Figure 5 we have attempted the coordination of the chromosome bands of D. subobscura and D. melanogaster in the section around the Mdh locus, which has been localized cytologically to the subdivision 3 1 B-F in D.melanogaster (VOELKERet al. 1978). In both species the Mdh gene is found in a chromosomal region characterized by a gooseneck with rather faint bands. In both species there is no comparable pattern found along the chromosomes U and 2L, respectively, implying that the concordance cannot be random. Yet, the cytological technique applied in this study is rather crude, and certainly not all bands become visible or discernible. SAURAand SORSA(1 979) have analyzed this region in electron micrographs of thin-sec- tioned chromosomes. They were able to identify as many as 38 bands in division 3 1 of D. melanogaster. In D. subobscura the number of bands estimated from light microscopic investigations is probably comparably high, but exact data are not available yet. With the introduction of less laborious techniques like the method of chromosome spreading (KALISCH and WHITMORE1983), however, an equivalent resolution of the banding pattern of D. subobscura should be possible in the near future.

The work was supported by the Deutsche Forschungsgemeinschaft (Projekt Sp 146/6-1). We are very grateful to I. KAIPF and K. STOCERERfor their excellent technical help, to J. ADAMS (Ann Arbor, Michigan) for critial discussion and to CH. REHMfor typing the manuscript. 924 W. PINSKER AND D. SPERLICH

D. sub D.mel U 2L

H 30 ,-- -

31 e------I -). 32

& 33

.c centromere

FIGURE 5.-Comparison of the banding patterns of the regions of the polytene chrombsomes containing the Mdh genes of D. subobscura and D. mclanogasfer. CYTOGENETIC MAPPING IN DROSOPHILA 925

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Corresponding editor: D. L. HARTL