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Uneven distribution of cloned transcribed DNA sequences in polytene chromosomes of Drosophila melanogaster
Cell Differentiation, 18 (1986) 145-149 Elsevier Scientific Publishers Ireland, Ltd.
145
C D F 00350
Uneven distribution of cloned transcribed D N A sequences in polytene chromosomes of Drosophilamelanogaster A.M. K o l c h i n s k y 1 R.P. V a s h a k i d z e 1, E.Yu. K u p e r t 2 a n d L.I. K o r o c h k i n 2 I Institute of Molecular Biology and -' Institute of Developmental Biolog~v, USSR Academy oJ Sciences~ Moscow, U. S~S. R. (Accepted 25 September 1985)
DNA fragments actively transcribed at different developmental stages of Drosophila melanogaster were cloned. In situ hybridization was used to study their distribution in the polytene chromosomes. The clones were dot-hybridized to poly(A) + RNA isolated at different developmental stages. Fifty-four of 67 clones were unique, 37 of them were localized. An irregular distribution of the cloned sequences was revealed. Twenty-three clones are localized in chromosome 3, six clones in the X-chromosome, and only eight in chromosome 2. There are transcriptionally active regions where, on a relatively small area of the chromosomes, a number of clones are localized (sections 18-19, 69-71, 82-85, 95-97). RNAs transcribed from the cloned sequences code for about 0.005 to 0.2% of the total poly(A) + RNA. mRNA; developmental regulation; in situ localization
Introduction
The uneven gene distribution in the Drosophila genome attracts much attention. The reasons for this phenomenon are still unknown. In some cases, gene clusters in certain chromosomes are related to a coordinated gene expression. Thus, multiple genes for ribosomal RNA, 5S RNA genes, histone genes, chorionic protein genes and genes for low molecular weight heat-shock proteins are grouped in one or several chromosomal regions. On the other hand, the tRNA genes, heat-shock protein genes and others are dispersed all over the genome (Doane and Treat-Clemons, 1982). This paper deals with the uneven distribution of randomly cloned D N A sequences, both those expressed uniformly at all six developmental stages of Drosophila and those predominantly transcribed only at some of these stages. An attempt
has been made to analyze this problem by a combination of cytological and genetic engineering techniques.
Materials and Methods
In the Drosophila melanogaster chromosomes, in situ hybridization was utilized to localize several genes intensively transcribed at different developmental stages. The use of cloned fragments of the genomic D N A has significant advantages in comparison with R N A or cloned c D N A hybridization. In particular, it allows the study of the most interesting clones directly. Besides, the preparation of c D N A involves selective synthesis and cloning of some sequences and deletion of others, and the labelling of R N A leads to the problem of widely differing concentrations of R N A species. There-
0045-6039/86/$03.50 ':' 1986 Elsevier Scientific Publishers Ireland, Ltd.
146 fore, we first o b t a i n e d a library of D N A fragments from D. melanogaster embryos cloned in pBR322 plasmid as described elsewhere (Kolchinsky et al., 1980). Then we selected clones which hybridize to in vitro ~:SI-labeled polyadenylated RNA, isolated from all six developmental stages, i.e. from the embryos, 1st, 2nd, and 3rd instar larvae, pupae and imago. The D N A of these selected clones were labelled with 3H-nucleotides by nick-translation and hybridized to polytene chromosomes in situ as described by Pardue and Gall (1975). In addition to in situ hybridization for characterizing the transcription time of cloned D N A fragments and their r e d u n d a n c y in the genome, we used dot-hybridization (Kafatos et al., 1979). The sites of hybridization were identified according to Bridges' map (1935) as presented by Lefevre (1976).
Results and Discussion The in situ localization of cloned fragments is listed in Table I. The approximate length of insertion is also indicated. Only the results of high quality hybridization are included in this table. In total, we studied 67 clones, of which 54 (80%) were ' u n i q u e ' , i.e., they were represented in the genome by no more than three to five copies. The n u m b e r of copies was estimated by dot-hybridization with total 32P-labelled D N A from embryos and in some cases verified using hybridization of 3-~P-clones with genomic Southern blots. Thirty-seven clones were localized in polytene chromosomes. Fig. 1 shows examples of in situ hybridizations, and Fig. 2 summarizes the localization of unique clones. The irregular distribution of these cloned D N A fragments in the D. melanogaster genome should be noted: 23 clones are localized in chromosome 3 (17 in 3R and six in 3L), six clones in the X-chromosome, and only eight in c h r o m o s o m e 2 (six in 2R and two in 2L). T r a n scriptionally active regions are established where several genes are localized. For example, in zone 97 of chromosome 3R, four clones can be found, in the zone 82-85 of chromosome 3R, eight clones, in the zone 69-71 of chromosome 3L, four clones, and in the zone 1 8 - 1 9 of the X-chromosome, four clones. Thus, the loci transcribed most actively at
TABLE I Cloned DNA fragments coding for abundant mRNAs: their in situ localization and approximate length Clone designation Dm A36 Dm 713 Dm 61 Dm 29 Dm 513 Dm 87 Dm 313 Dm A52 Dm A60 Dm 32 Dm A98 Dm A61 Dm A48 Din A92 Dm AI6 Dm AI00 Dm A41 Dm A85 Dm 613 Dm A38 Dm A96 Dm A90 Dm A46 Dm 21 Dm A83 Dm 16 Dm 89 Dm A49 Dm Alll Dm A81 Dm 22 Dm Al13 Dm 62 Dm 314 Dm 23 Dm 210 Dm 28
In situ localization 3C3 I4D 18C ISEF 18F-19A 19A 25E 25F 43C 53D1.2 54E 56F1 58EF 60C 63D 69D1-E1 69F 71BC 71 F-72A 75F 82BI,2 82D 83BC 83CD 83F-84A 84D 84D 85B1,2 87B 89B1-4 95E 95E 97A 97B 97C 97C-D 99F
Approximate insertion size" 12 15 1.9 5.1 5.0 15 6.6 1.1 9.5 15 5.8 15 5.6 15 6.7 6.0 8.6 5.2 7.6 14 6.4 8.7 12 7.3 7.0 15 10 4.7 2.1 15 4.1 3.2 2.5 1.4 2.8 2.5 0.8
" Main length of insert is ca. 7 kb.
different developmental stages are localized pred o m i n a n t l y in several regions of chromosome 3. It is noteworthy that 82 to 85 regions c o n t a i n a n u m b e r of homeosis m u t a t i o n s (Lindsley and Grell, 1968), and a n u m b e r of puffs develop here in the salivary glands of the 3rd instar larvae and of pre-pupae. W h e n studying the transcription time of these clones, we found that almost all of them
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were highly active at the pupal stage. Judging by appearance, this region is of great importance for the development of imaginal organs, and it is the place where the genes coding for rich m R N A at the corresponding stages are concentrated. In contrast to many attempts to clone D N A coding for abundant polyadenylated RNAs in cultured cells (see, for instance, Ilyin et al., 1978), our approach produced mostly unique D N A fragments. Only about 20% of all clones are repeated 10-100 times in the genome and three of them show unstable localization in the polytene chromosomes upon in situ hybridization. In order to study the expression of cloned D N A fragments, we used polyadenylated RNA from whole animals at different developmental stages. R N A preparations were labelled with polynucleotide kinase in the presence of [32P]ATP following mild alkaline degradation and dot-hybridized to plasmids containing the cloned fragments. When this approach was used, the steady state amount of transcripts was assessed by the intensity of corresponding spots on X-ray photographs. For comparison, filters were mounted with the cloned D N A fragments coding for a known amount of R N A (small fragments of ribosomal genes, histone genes, 5S RNA genes were applied as dots on the same filters). We estimated the amount of more abundant polyadenylated RNA to equal 0.1-0.3%, while the weakest spots on the radioautographs bound 10-50 times less 3zP-labelled RNA. The results of these experiments are summarized in Table II. Unique clones are expressed more or less regularly at all stages, six of them
T A B L E It Expression of cloned
unique fragments at
different
melanogaster developmental stages N u m b e r of stages showing significant levels of transcripts from a given clone
N u m b e r of unique clones
1
4
2 3 4 5 6
3 13 10 13 11
D.
X
2L
2R 43
3L 63
2.~
3R 82 83 84 85 87
69
89
71
14
53 54 75
56
I~
,58. 60;
95 97: 99
Fig. 2. A scheme of distribution of the hybridization regions on the salivaryglands polytene chromosomesDNA to the isolated and identified clones. Numerals: chromosome regions. being localized in chromosome 3. Only four clones hybridized to RNA predominantly at one of the developmental stages. This suggests that a restricted number of cloned fragments provide stage-specific transcription. Many D N A fragments are transcribed over an interval, e.g. clone 513 (locus 1-18F-19A) is transcribed intensively in the embryos, 1st instar larvae and pupae, but minimally in the 3rd instar larvae. During embryogenesis a maximal number of cloned DNAs is being transcribed. Since RNA was isolated from whole animals, two types of abundant RNAs can be represented in these preparations. First, the 'luxury' RNA which accumulate in certain differentiated tissues during certain periods, and second, 'house-keeping' RNA, relatively abundantly represented in all cells. Since most of our clones are expressed at two or more developmental stages, they seem to represent ' house-keeping' genes. We hope that the finding of the transcription schedule as well as the estimation of the level of transcripts in different tissues will allow identification of some cloned sequences. References Bridges, C.B.: Salivary chromosome maps, with a key to the banding of the chromosomes of Drosophila melanogaster. ,1. Heredity 26, 60-64 (1935).
149 Doane, W. and L. Treat-Clemons: Biochemical loci of the "' fruit fly" (Drosophila melanogaster). Drosophila Inf. Serv. 58, 41-59 (1982). ilyin, Y., N. Tchurikov, E. Ananiev, A. Ryskov, G. Yenikolopov, S. Limborska, N. Maleeva, V. Gvozdev and G. Georgiev: Studies on the D N A fragments of m a m m a l s and Drosophila containing structural genes and adjacent sequences. Cold Spring Harbor Symp. Quant. Biol. 42, 959-969 (1978). Kafatos, F., C. Jones and A. Efstratiadis: Determination of nucleic acids sequence homologies and relative concentrations by a dot-hybridization procedure. Nucleic Acids Res. 7, 1541-1552 (1979). Kolchinsky, A., R. Vashakidze and A. Mirzabekov: A simple
method of cloning eukaryotic DNA. Ribosomal genes from Drosophila containing new insertions in the 26S gene. Mol. Biol. (USSR) 14, 1098-1109 (1980). Lefevre, G.: A photographic representation and interpretation of the polytene chromosomes of Drosophila melanogaster salivary glands. In: The Genetics and Biology of Drosophila, Vol. la, pp. 31-36, ed. M. Ashburner (Academic Press, New York) (1976). Lindsley, D. and E. Grell: Genetic variations of D. melanogaster. Carnegie Inst. Washington, Publ. No. 627 (1968). Pardue, M.L. and J.G. Gall: Nucleic acid hybridization to the D N A of cytological preparations. In: Methods in Cell Biology, Vol. 10, ed. D.M. Prescott (Academic Press, New York) pp. 1 - 6 (1975).
145
C D F 00350
Uneven distribution of cloned transcribed D N A sequences in polytene chromosomes of Drosophilamelanogaster A.M. K o l c h i n s k y 1 R.P. V a s h a k i d z e 1, E.Yu. K u p e r t 2 a n d L.I. K o r o c h k i n 2 I Institute of Molecular Biology and -' Institute of Developmental Biolog~v, USSR Academy oJ Sciences~ Moscow, U. S~S. R. (Accepted 25 September 1985)
DNA fragments actively transcribed at different developmental stages of Drosophila melanogaster were cloned. In situ hybridization was used to study their distribution in the polytene chromosomes. The clones were dot-hybridized to poly(A) + RNA isolated at different developmental stages. Fifty-four of 67 clones were unique, 37 of them were localized. An irregular distribution of the cloned sequences was revealed. Twenty-three clones are localized in chromosome 3, six clones in the X-chromosome, and only eight in chromosome 2. There are transcriptionally active regions where, on a relatively small area of the chromosomes, a number of clones are localized (sections 18-19, 69-71, 82-85, 95-97). RNAs transcribed from the cloned sequences code for about 0.005 to 0.2% of the total poly(A) + RNA. mRNA; developmental regulation; in situ localization
Introduction
The uneven gene distribution in the Drosophila genome attracts much attention. The reasons for this phenomenon are still unknown. In some cases, gene clusters in certain chromosomes are related to a coordinated gene expression. Thus, multiple genes for ribosomal RNA, 5S RNA genes, histone genes, chorionic protein genes and genes for low molecular weight heat-shock proteins are grouped in one or several chromosomal regions. On the other hand, the tRNA genes, heat-shock protein genes and others are dispersed all over the genome (Doane and Treat-Clemons, 1982). This paper deals with the uneven distribution of randomly cloned D N A sequences, both those expressed uniformly at all six developmental stages of Drosophila and those predominantly transcribed only at some of these stages. An attempt
has been made to analyze this problem by a combination of cytological and genetic engineering techniques.
Materials and Methods
In the Drosophila melanogaster chromosomes, in situ hybridization was utilized to localize several genes intensively transcribed at different developmental stages. The use of cloned fragments of the genomic D N A has significant advantages in comparison with R N A or cloned c D N A hybridization. In particular, it allows the study of the most interesting clones directly. Besides, the preparation of c D N A involves selective synthesis and cloning of some sequences and deletion of others, and the labelling of R N A leads to the problem of widely differing concentrations of R N A species. There-
0045-6039/86/$03.50 ':' 1986 Elsevier Scientific Publishers Ireland, Ltd.
146 fore, we first o b t a i n e d a library of D N A fragments from D. melanogaster embryos cloned in pBR322 plasmid as described elsewhere (Kolchinsky et al., 1980). Then we selected clones which hybridize to in vitro ~:SI-labeled polyadenylated RNA, isolated from all six developmental stages, i.e. from the embryos, 1st, 2nd, and 3rd instar larvae, pupae and imago. The D N A of these selected clones were labelled with 3H-nucleotides by nick-translation and hybridized to polytene chromosomes in situ as described by Pardue and Gall (1975). In addition to in situ hybridization for characterizing the transcription time of cloned D N A fragments and their r e d u n d a n c y in the genome, we used dot-hybridization (Kafatos et al., 1979). The sites of hybridization were identified according to Bridges' map (1935) as presented by Lefevre (1976).
Results and Discussion The in situ localization of cloned fragments is listed in Table I. The approximate length of insertion is also indicated. Only the results of high quality hybridization are included in this table. In total, we studied 67 clones, of which 54 (80%) were ' u n i q u e ' , i.e., they were represented in the genome by no more than three to five copies. The n u m b e r of copies was estimated by dot-hybridization with total 32P-labelled D N A from embryos and in some cases verified using hybridization of 3-~P-clones with genomic Southern blots. Thirty-seven clones were localized in polytene chromosomes. Fig. 1 shows examples of in situ hybridizations, and Fig. 2 summarizes the localization of unique clones. The irregular distribution of these cloned D N A fragments in the D. melanogaster genome should be noted: 23 clones are localized in chromosome 3 (17 in 3R and six in 3L), six clones in the X-chromosome, and only eight in c h r o m o s o m e 2 (six in 2R and two in 2L). T r a n scriptionally active regions are established where several genes are localized. For example, in zone 97 of chromosome 3R, four clones can be found, in the zone 82-85 of chromosome 3R, eight clones, in the zone 69-71 of chromosome 3L, four clones, and in the zone 1 8 - 1 9 of the X-chromosome, four clones. Thus, the loci transcribed most actively at
TABLE I Cloned DNA fragments coding for abundant mRNAs: their in situ localization and approximate length Clone designation Dm A36 Dm 713 Dm 61 Dm 29 Dm 513 Dm 87 Dm 313 Dm A52 Dm A60 Dm 32 Dm A98 Dm A61 Dm A48 Din A92 Dm AI6 Dm AI00 Dm A41 Dm A85 Dm 613 Dm A38 Dm A96 Dm A90 Dm A46 Dm 21 Dm A83 Dm 16 Dm 89 Dm A49 Dm Alll Dm A81 Dm 22 Dm Al13 Dm 62 Dm 314 Dm 23 Dm 210 Dm 28
In situ localization 3C3 I4D 18C ISEF 18F-19A 19A 25E 25F 43C 53D1.2 54E 56F1 58EF 60C 63D 69D1-E1 69F 71BC 71 F-72A 75F 82BI,2 82D 83BC 83CD 83F-84A 84D 84D 85B1,2 87B 89B1-4 95E 95E 97A 97B 97C 97C-D 99F
Approximate insertion size" 12 15 1.9 5.1 5.0 15 6.6 1.1 9.5 15 5.8 15 5.6 15 6.7 6.0 8.6 5.2 7.6 14 6.4 8.7 12 7.3 7.0 15 10 4.7 2.1 15 4.1 3.2 2.5 1.4 2.8 2.5 0.8
" Main length of insert is ca. 7 kb.
different developmental stages are localized pred o m i n a n t l y in several regions of chromosome 3. It is noteworthy that 82 to 85 regions c o n t a i n a n u m b e r of homeosis m u t a t i o n s (Lindsley and Grell, 1968), and a n u m b e r of puffs develop here in the salivary glands of the 3rd instar larvae and of pre-pupae. W h e n studying the transcription time of these clones, we found that almost all of them
3'
CD
3
©
~J
©
C~
e~
Cri
t
J
148
were highly active at the pupal stage. Judging by appearance, this region is of great importance for the development of imaginal organs, and it is the place where the genes coding for rich m R N A at the corresponding stages are concentrated. In contrast to many attempts to clone D N A coding for abundant polyadenylated RNAs in cultured cells (see, for instance, Ilyin et al., 1978), our approach produced mostly unique D N A fragments. Only about 20% of all clones are repeated 10-100 times in the genome and three of them show unstable localization in the polytene chromosomes upon in situ hybridization. In order to study the expression of cloned D N A fragments, we used polyadenylated RNA from whole animals at different developmental stages. R N A preparations were labelled with polynucleotide kinase in the presence of [32P]ATP following mild alkaline degradation and dot-hybridized to plasmids containing the cloned fragments. When this approach was used, the steady state amount of transcripts was assessed by the intensity of corresponding spots on X-ray photographs. For comparison, filters were mounted with the cloned D N A fragments coding for a known amount of R N A (small fragments of ribosomal genes, histone genes, 5S RNA genes were applied as dots on the same filters). We estimated the amount of more abundant polyadenylated RNA to equal 0.1-0.3%, while the weakest spots on the radioautographs bound 10-50 times less 3zP-labelled RNA. The results of these experiments are summarized in Table II. Unique clones are expressed more or less regularly at all stages, six of them
T A B L E It Expression of cloned
unique fragments at
different
melanogaster developmental stages N u m b e r of stages showing significant levels of transcripts from a given clone
N u m b e r of unique clones
1
4
2 3 4 5 6
3 13 10 13 11
D.
X
2L
2R 43
3L 63
2.~
3R 82 83 84 85 87
69
89
71
14
53 54 75
56
I~
,58. 60;
95 97: 99
Fig. 2. A scheme of distribution of the hybridization regions on the salivaryglands polytene chromosomesDNA to the isolated and identified clones. Numerals: chromosome regions. being localized in chromosome 3. Only four clones hybridized to RNA predominantly at one of the developmental stages. This suggests that a restricted number of cloned fragments provide stage-specific transcription. Many D N A fragments are transcribed over an interval, e.g. clone 513 (locus 1-18F-19A) is transcribed intensively in the embryos, 1st instar larvae and pupae, but minimally in the 3rd instar larvae. During embryogenesis a maximal number of cloned DNAs is being transcribed. Since RNA was isolated from whole animals, two types of abundant RNAs can be represented in these preparations. First, the 'luxury' RNA which accumulate in certain differentiated tissues during certain periods, and second, 'house-keeping' RNA, relatively abundantly represented in all cells. Since most of our clones are expressed at two or more developmental stages, they seem to represent ' house-keeping' genes. We hope that the finding of the transcription schedule as well as the estimation of the level of transcripts in different tissues will allow identification of some cloned sequences. References Bridges, C.B.: Salivary chromosome maps, with a key to the banding of the chromosomes of Drosophila melanogaster. ,1. Heredity 26, 60-64 (1935).
149 Doane, W. and L. Treat-Clemons: Biochemical loci of the "' fruit fly" (Drosophila melanogaster). Drosophila Inf. Serv. 58, 41-59 (1982). ilyin, Y., N. Tchurikov, E. Ananiev, A. Ryskov, G. Yenikolopov, S. Limborska, N. Maleeva, V. Gvozdev and G. Georgiev: Studies on the D N A fragments of m a m m a l s and Drosophila containing structural genes and adjacent sequences. Cold Spring Harbor Symp. Quant. Biol. 42, 959-969 (1978). Kafatos, F., C. Jones and A. Efstratiadis: Determination of nucleic acids sequence homologies and relative concentrations by a dot-hybridization procedure. Nucleic Acids Res. 7, 1541-1552 (1979). Kolchinsky, A., R. Vashakidze and A. Mirzabekov: A simple
method of cloning eukaryotic DNA. Ribosomal genes from Drosophila containing new insertions in the 26S gene. Mol. Biol. (USSR) 14, 1098-1109 (1980). Lefevre, G.: A photographic representation and interpretation of the polytene chromosomes of Drosophila melanogaster salivary glands. In: The Genetics and Biology of Drosophila, Vol. la, pp. 31-36, ed. M. Ashburner (Academic Press, New York) (1976). Lindsley, D. and E. Grell: Genetic variations of D. melanogaster. Carnegie Inst. Washington, Publ. No. 627 (1968). Pardue, M.L. and J.G. Gall: Nucleic acid hybridization to the D N A of cytological preparations. In: Methods in Cell Biology, Vol. 10, ed. D.M. Prescott (Academic Press, New York) pp. 1 - 6 (1975).