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Visualization of the Silk Fibroin Transcription Unit and Nascent Silk Fibroin Molecules on Polyribosomes of Bombyx mori
Visualization of the Silk Fibroin Transcription Unit and Nascent Silk Fibroin Molecules on Polyribosomes of Bombyx mori
~
STEVEN L. MCKNICHT, NELDAL. SULLIVAN AND OSC4R
L. MILLER,JR.
Department of Biology Uniuenity of Virginia ClzarlottesuiUe, Virginia
The unique physical properties of the silk fibroin gene of Bornbyx mori, its complementary mRNA molecule and the fibroin polypeptide have allowed biochemical probes to define the kinetics of fibroin production. Late in larval development, the highly polyploid posterior silk gland cells contribute over 80%of their protei i synthesis to the production of silk fibroin ( 1 ) . By modifying chromosome spreading techniques first adapted for visualizing extrachromosomel nucleolar genes of amphibian oocytes ( 2 ), we have examined the transcriptional ( McKnight ) and translational ( Sullivan) organization of silk producing Bornbyr cells. To spread the chromosomes, 5 mg of the gland tissue were dispersed with jewelers’ forceps in 3 nil of a 0.05%Joy detergent solution adjusted to pH 8.5 with NaOH-borate buffer. The suspewion was then centrifuged through a formalin-sucrose cushion onto a carbon-coated electron microscope grid and prepared for transmission electron microscopy by techniques previously reported ( 3 ) . Adequately dispersed Bornbyx posterior silk-gland genomcs show: ( a ) inactive “ b e a d e d chromatin regions; ( b ) active ribosomal genes ( Fig. l a ) ; ( c ) variously sized non-nucleolar genes that are typically populated with low densities of RNA polymerase molecules; and ( d ) a distinct population of ribonucleoprotein fibril gradients between 5 and 6 p n ~long that are packed with almost as many RNA polymerases per unit length as the rRNA genes, but are not present in tandem array (Figs. lb, l c and 2). The distribution of the three categories of ribonucleoprotein fibril gradients changes as larvae proceed through the fifth instar, with the category of long, polymerase-dense gradients becoming more prominent. We identify these long, polymerase-dense gradients as active silk fibroin genes for the following reasons. First, the mean length of these 313
VISUALIZATION OF TRANSCRIPTION UNITS
315
distinct transcription units is 5.43 k 0.24 pm ( N = 14). If the B-conformation length of the gene is foreshortened by the amount we have estimated for rRNA genes (-12%), the obscrved length would correspond to a tcmplate slightly longer than 1.8 x 10' base-pairs, a length that is very close to the gene size estimated by biochemical probes (-17,000 base-pairs; 4 ) . Second, preliminary observations indicate that the middle portion of the silk gland, which synthesizes little or no fibroin mRNA, does not contain the very long polymerase-dense gradients observed in the posterior portion. Third, the -5.4 p m gridients are not tandemly repeated, which is consistent with data showin; the fibroin gene to exist only once per haploid genornc equivalent ( 5 ) .And fourth, these gradients are present on loci that are essentially space-filled with RNA polymerases. Analysis of fibroin mRNA production in the fifth larval instar indicates that such single-copy genes would have to be loaded with polymerases in order to account for thc large number of messages synthesized per unit time during this stage of development (Y. Suzuki, personal communication). In most instances, the terminal regions of the fibroin transcription units are obscured because of the extraordinary length of the more terminal ribonucleoprotein fibrils, and the prestmce of overlapping chromatin strands. In one case, howevcr, wt' have observed that a portion of the most distal fibrils appear to have been cleaved (Figs. l b and l c ) . This may represent primary transcript processirig, and, if so, would indicate that the fibroin gene produces a short-lived precursor molecule somewhat larger than the fibroin mRNA (see Lizardi, page 301). Similar ribonucleoprotein fibril processing has been reported by Laird and Chooi ( 6 ) which suggests that cleavage of nascent ribonucleoprotein molecules occurs on the nurse-cell genome of Drosophilu melunogaster. The polyribosomes of posterior silk-gland cdls were inspected using methods similar to those described for genome pieparation except that the FIG. l a . Electron micrograph of Bombyx mori ribosomal ribonu'cleoprotein matrices. The sample was prepared from mid-5th instar posterior silk gland tissue. FICS.l b and lc. Electron Micrograph of a putative silk fibroin transcription unit. The sample was prepared from mid-5th instar posterior silk gland tissue. Arrows in 1L point to sites of initiation ( i ) and termination ( t ) of transcription. Contour length ( i ) * ( t ) measures -5.8 pxn. Figure l c is a tracing of Fig. l b and shows the putative endnucleolytic cleavage site (large arrow). Of the (imost terminal nascent transcripts, 5 appear cleaved (small arrows) and have a mean length of 0.19 pm, which is just under one-fifth the mean length ( 1.02 pm) of t t e most terminal unprocessed transcript$. Endonucleolytic cleavage site ( ecs ) occurs -4.8 pm from initiation site ( i ) , thus accounting for slighty less than four-fifths of t.he full gradient length. The density of RNA polymerases hound near the terminus of the gradient appears less than that in the more proximal regions.
FIG. 2. Electron iiiicrograph of a putative silk fibroin transcription unit. The sample W'IS prepared from mid-5th instar posterior silk gland tissue. Arrows point to sites of initiation ( i ) and termination ( t ) of transcription. Contour length ( i ) +( t ) measures -5.3 pm. The template is complexed with -40 RNA polymerase molecules per micrometer of contour length.
316
FIGS.3a, 3b, and 3c. Electron micrographs of polysomes isolated from the late 5th instar of posterior silk gland cells. The polysomes shown in Figs. 3a and 3b were isolated as described in the text, except that no cyclohevimide was used. Translational polarity is indicated by 5’ and 3’ symbols. Arrows point to putative nascent silk fibroin polypeptides. Bars represent 0.1 pm. 317
318
STEVEN L. hlCKNIGHT ET. AI..
tissue was suspended in a solution of cyclohexiinide (20 pglml) for up to 24 hours prior to homogenization. The large majority of the polysomes observed after this treatment (Fig. 3 ) exhibit an array of extended thin fibrils singly attached to individual ribosomes. The attached fibrils reach a maximum length of -0.1 pin and have a distinctive beaded appearance. Tangling and shearing of these very long polysomes has so far prevented an accurate determination of their sizr, but the range probably lies between 50 and 80 ribosomes. The attached fibrils are identified as nascent silk fibroin polypeptides because: ( a ) the late fifth instar posterior silk-gland cells synthesize almost exclusively silk fibroin; ( b ) they generate gradients of increasing length along the polysome as expected from the known mechanism of mRNA translation, and establish its polarity; ( c ) the contents of the lumen of the posterior portion of the silk gland, dispersed in the same way, consists priniarily of molecules having essentially the same distinctive morphology and sizc as the longest fibrils found at the 3’ end of the polyribosomes; and ( d ) the extreme size of the silk fibroin molecule [estimated to range between 170,000 daltons ( 7 ) , and 370,000 daltons, ( 8) ] requires polysomes in this size range. Visualization of thcse nascent fibroin polypeptides is possible both because of their size and because they contain repeating amino-acid sequences that take the form of folded antiparallel ,&pleated sheets that extend in a linear, rather than a globular, conformation. To our knowledge, these observations mark the first time that a specific structural eukaryotic gene has been visualized and the first visual confirmation of the accepted biochemical interpretation of protein synthesis.
ACKNOWLEDGMENTS We thank Ur. D. D. Brown for his suggestion that we initiate this study, and are grateful to Dr. Y. Suzuki and Paul Giza for generously supplying us with Bomhyx mori embryos and larvae. We acknowledge and appreciate the communication of unpublished data and stimulating discussion provided by Urs. Suzuki, Brown, and P a d Lizardi. We also thank Ms L. Blanks for her excellent technical assistance. Supported by NSF Grant BMS73-01131-AOl and USPHS-NIGMS Grant 1 R01 GM21020-01.
REFERENCES 1. Y. Tashiro, T. Morinioto, S. Matsnura and S. Nagata, J. Cell Biol. 38, 574 ( 1968). 2 . 0. L. Miller, Jr. and B. R. Beatty, Science 164, 955 (19G9). 3. S. L. McKnight and 0. L. Miller, Jr., Cell 8, 305 (1976). 4. P. M. Lizardi and D. D. Brown, Cell 4, 207-215 (1975). 5 . P. M. Lizardi and D. 1).Brown, CSHSQB 38, 701 (1973). 6. C . D. Laird and W. Y. Chooi, Cl~romosomo,in press (1976). 7 . Y. Tashiro and E. Otsuki, BBA 214,265 (1970). 8. K. U. Sprague, Bchetn 14, 925 ( 1975).
~
STEVEN L. MCKNICHT, NELDAL. SULLIVAN AND OSC4R
L. MILLER,JR.
Department of Biology Uniuenity of Virginia ClzarlottesuiUe, Virginia
The unique physical properties of the silk fibroin gene of Bornbyx mori, its complementary mRNA molecule and the fibroin polypeptide have allowed biochemical probes to define the kinetics of fibroin production. Late in larval development, the highly polyploid posterior silk gland cells contribute over 80%of their protei i synthesis to the production of silk fibroin ( 1 ) . By modifying chromosome spreading techniques first adapted for visualizing extrachromosomel nucleolar genes of amphibian oocytes ( 2 ), we have examined the transcriptional ( McKnight ) and translational ( Sullivan) organization of silk producing Bornbyr cells. To spread the chromosomes, 5 mg of the gland tissue were dispersed with jewelers’ forceps in 3 nil of a 0.05%Joy detergent solution adjusted to pH 8.5 with NaOH-borate buffer. The suspewion was then centrifuged through a formalin-sucrose cushion onto a carbon-coated electron microscope grid and prepared for transmission electron microscopy by techniques previously reported ( 3 ) . Adequately dispersed Bornbyx posterior silk-gland genomcs show: ( a ) inactive “ b e a d e d chromatin regions; ( b ) active ribosomal genes ( Fig. l a ) ; ( c ) variously sized non-nucleolar genes that are typically populated with low densities of RNA polymerase molecules; and ( d ) a distinct population of ribonucleoprotein fibril gradients between 5 and 6 p n ~long that are packed with almost as many RNA polymerases per unit length as the rRNA genes, but are not present in tandem array (Figs. lb, l c and 2). The distribution of the three categories of ribonucleoprotein fibril gradients changes as larvae proceed through the fifth instar, with the category of long, polymerase-dense gradients becoming more prominent. We identify these long, polymerase-dense gradients as active silk fibroin genes for the following reasons. First, the mean length of these 313
VISUALIZATION OF TRANSCRIPTION UNITS
315
distinct transcription units is 5.43 k 0.24 pm ( N = 14). If the B-conformation length of the gene is foreshortened by the amount we have estimated for rRNA genes (-12%), the obscrved length would correspond to a tcmplate slightly longer than 1.8 x 10' base-pairs, a length that is very close to the gene size estimated by biochemical probes (-17,000 base-pairs; 4 ) . Second, preliminary observations indicate that the middle portion of the silk gland, which synthesizes little or no fibroin mRNA, does not contain the very long polymerase-dense gradients observed in the posterior portion. Third, the -5.4 p m gridients are not tandemly repeated, which is consistent with data showin; the fibroin gene to exist only once per haploid genornc equivalent ( 5 ) .And fourth, these gradients are present on loci that are essentially space-filled with RNA polymerases. Analysis of fibroin mRNA production in the fifth larval instar indicates that such single-copy genes would have to be loaded with polymerases in order to account for thc large number of messages synthesized per unit time during this stage of development (Y. Suzuki, personal communication). In most instances, the terminal regions of the fibroin transcription units are obscured because of the extraordinary length of the more terminal ribonucleoprotein fibrils, and the prestmce of overlapping chromatin strands. In one case, howevcr, wt' have observed that a portion of the most distal fibrils appear to have been cleaved (Figs. l b and l c ) . This may represent primary transcript processirig, and, if so, would indicate that the fibroin gene produces a short-lived precursor molecule somewhat larger than the fibroin mRNA (see Lizardi, page 301). Similar ribonucleoprotein fibril processing has been reported by Laird and Chooi ( 6 ) which suggests that cleavage of nascent ribonucleoprotein molecules occurs on the nurse-cell genome of Drosophilu melunogaster. The polyribosomes of posterior silk-gland cdls were inspected using methods similar to those described for genome pieparation except that the FIG. l a . Electron micrograph of Bombyx mori ribosomal ribonu'cleoprotein matrices. The sample was prepared from mid-5th instar posterior silk gland tissue. FICS.l b and lc. Electron Micrograph of a putative silk fibroin transcription unit. The sample was prepared from mid-5th instar posterior silk gland tissue. Arrows in 1L point to sites of initiation ( i ) and termination ( t ) of transcription. Contour length ( i ) * ( t ) measures -5.8 pxn. Figure l c is a tracing of Fig. l b and shows the putative endnucleolytic cleavage site (large arrow). Of the (imost terminal nascent transcripts, 5 appear cleaved (small arrows) and have a mean length of 0.19 pm, which is just under one-fifth the mean length ( 1.02 pm) of t t e most terminal unprocessed transcript$. Endonucleolytic cleavage site ( ecs ) occurs -4.8 pm from initiation site ( i ) , thus accounting for slighty less than four-fifths of t.he full gradient length. The density of RNA polymerases hound near the terminus of the gradient appears less than that in the more proximal regions.
FIG. 2. Electron iiiicrograph of a putative silk fibroin transcription unit. The sample W'IS prepared from mid-5th instar posterior silk gland tissue. Arrows point to sites of initiation ( i ) and termination ( t ) of transcription. Contour length ( i ) +( t ) measures -5.3 pm. The template is complexed with -40 RNA polymerase molecules per micrometer of contour length.
316
FIGS.3a, 3b, and 3c. Electron micrographs of polysomes isolated from the late 5th instar of posterior silk gland cells. The polysomes shown in Figs. 3a and 3b were isolated as described in the text, except that no cyclohevimide was used. Translational polarity is indicated by 5’ and 3’ symbols. Arrows point to putative nascent silk fibroin polypeptides. Bars represent 0.1 pm. 317
318
STEVEN L. hlCKNIGHT ET. AI..
tissue was suspended in a solution of cyclohexiinide (20 pglml) for up to 24 hours prior to homogenization. The large majority of the polysomes observed after this treatment (Fig. 3 ) exhibit an array of extended thin fibrils singly attached to individual ribosomes. The attached fibrils reach a maximum length of -0.1 pin and have a distinctive beaded appearance. Tangling and shearing of these very long polysomes has so far prevented an accurate determination of their sizr, but the range probably lies between 50 and 80 ribosomes. The attached fibrils are identified as nascent silk fibroin polypeptides because: ( a ) the late fifth instar posterior silk-gland cells synthesize almost exclusively silk fibroin; ( b ) they generate gradients of increasing length along the polysome as expected from the known mechanism of mRNA translation, and establish its polarity; ( c ) the contents of the lumen of the posterior portion of the silk gland, dispersed in the same way, consists priniarily of molecules having essentially the same distinctive morphology and sizc as the longest fibrils found at the 3’ end of the polyribosomes; and ( d ) the extreme size of the silk fibroin molecule [estimated to range between 170,000 daltons ( 7 ) , and 370,000 daltons, ( 8) ] requires polysomes in this size range. Visualization of thcse nascent fibroin polypeptides is possible both because of their size and because they contain repeating amino-acid sequences that take the form of folded antiparallel ,&pleated sheets that extend in a linear, rather than a globular, conformation. To our knowledge, these observations mark the first time that a specific structural eukaryotic gene has been visualized and the first visual confirmation of the accepted biochemical interpretation of protein synthesis.
ACKNOWLEDGMENTS We thank Ur. D. D. Brown for his suggestion that we initiate this study, and are grateful to Dr. Y. Suzuki and Paul Giza for generously supplying us with Bomhyx mori embryos and larvae. We acknowledge and appreciate the communication of unpublished data and stimulating discussion provided by Urs. Suzuki, Brown, and P a d Lizardi. We also thank Ms L. Blanks for her excellent technical assistance. Supported by NSF Grant BMS73-01131-AOl and USPHS-NIGMS Grant 1 R01 GM21020-01.
REFERENCES 1. Y. Tashiro, T. Morinioto, S. Matsnura and S. Nagata, J. Cell Biol. 38, 574 ( 1968). 2 . 0. L. Miller, Jr. and B. R. Beatty, Science 164, 955 (19G9). 3. S. L. McKnight and 0. L. Miller, Jr., Cell 8, 305 (1976). 4. P. M. Lizardi and D. D. Brown, Cell 4, 207-215 (1975). 5 . P. M. Lizardi and D. 1).Brown, CSHSQB 38, 701 (1973). 6. C . D. Laird and W. Y. Chooi, Cl~romosomo,in press (1976). 7 . Y. Tashiro and E. Otsuki, BBA 214,265 (1970). 8. K. U. Sprague, Bchetn 14, 925 ( 1975).