- Email: [email protected]
On the origin of labeled RNA in the cytoplasm of mitotic root tip cells of Vicia faba
178 ON THE
ORIGIN MITOTIC N. K. DAS,
Department
OF LABELED ROOT
TIP
ELSIE
of Zoology,
RNA CELLS
IN THE OF WCIA
P. SIEGEL
University
CYTOPLASM
OF
FABA
and M. ALFERT
of California,
Berkeley,
C&j’.,
U.S.A.
Received May 24, 1965
PULSE-LABELING experiments have shown that meta-anaphase cells of both plant and animal tissues are inactive with respect to RNA synthesis [2, 16, 181. Harris and LaCour [9], however, pointed out that pulse-labeling experiments may not be sensitive enough to detect low levels of cytoplasmic RNA synthesis in mitotic cells as well as in interphase cells. They observed the presence of labeled RNA in the cytoplasm of meta-anaphase cells of Vicia faba root tip meristems exposed to tritiated guanosine for 30 to 90 min. The duration of mitosis in this tissue had been assumed to be about 4 hr [7]. Since prophase occupies about one half of the total mitotic period and since RNA is not produced by the condensed meta-anaphase chromosomes they postulate that the observed cytoplasmic labeling is evidence of RNA synthesis in the cytoplasm during mitosis. In earlier [ll] and more recent [8] studies, however, the duration of mitosis in Vicia faba root meristem cells has been found to be about 2 rather than 4 hr. It seems very likely, therefore, that a considerable number of prophase cells, which are active in RNA synthesis, might have moved to meta-anaphase within 30 min, the earliest incorporation time used by Harris and LaCour. This possibility is tested in the present autoradiographic study. The gradual progression of cells to meta-anaphase has been followed by labeling with tritiated thymidine (see [19, 201) and the resulting data are compared with those obtained from the incorporation of tritium labeled RNA precursors. In experiments designed to determine the duration of the mitotic period, secondary roots of Vicia faba grown in aerated tap water were exposed to 1 +/ml of 3H-thymidine (spec. act. 3 and 6.7 c/miM’) for 30 min, washed thoroughly, and allowed to grow further in aerated tap water. Samples were collected from 0 to 10: hr. In other experiments, secondary roots were immersed in tap water with added 50 /+/ml of 3H-uridine (spec. act. 3.7 c/m&Z) or 50 ,+/ml of 3H-guanosine (spec. act. 1.5 cimM) for 10 to 90 min. All samples were fixed in acetic alcohol. Roots treated with 3Hthymidine were stained with Feulgen and squashed on slides. Since the hydrolysis in 1 N HCl used in the Feulgen staining procedure removes RNA, roots exposed to 3H-uridine or 3H-guanosine were softened by hydrolyzing in 10 per cent pectinase solution at pH 4 for 1 hr prior to squashing, or 2- 3 /A paraffin sections were prepared; these slides were then treated with cold TCA to remove acid soluble nucleotides. Autoradiographs were prepared by dipping slides in liquid emulsion (NTB2). Cells containing labeled RNA were stained with Harris’ hematoxylin through the processed emulsion. Some of the slides were digested with RNase, DNase, or hot TCA to remove This study was supported by grants from the National U.S. Public Health Service (RG-6025). Experimental
Cell Research 40
Science Foundation
(GB-1642)
and the
Ltrbeled RLYL4in cytoplasm of mitotic
cells
nucleic acids prior to coating slides with emulsion. A relatively long exposure of emulsion resulted in detection of some radioactivity, especially in the cytoplasm, that \yas not removable by nuclease or hot TCA digestion, suggesting that tritium is also incorporated into compounds other than nucleic acids (see also 15, 14, 171). Such non-specific labeling probably results from the small amount of impurities that is always present in commercially available labeled nucleosides [l]. Autoradiographic exposures (1-21 days) were chosen so that grains were due predominantly to the tritium incorporated into nucleic acids. The frequency of mitotic cells labeled 3H-uridine, or 3H-guanosine was determined from squashes with 3H-thymidinc, as well as from sections. A preponderance of labeled cells had grain densities many times greater than background. Cells were considered to be labeled, however, if the number of grains exceeded five above background. Grain counts (corrected for background) were also made over sectioned mitotic cells labeled with 3H-uridine. At about 2 hr after an initial 30-min exposure of roots to 3H-thymidine the first labeled prophases begin to appear and 4 hr later all prophases are labeled (Fig. 1). The first labeled meta-anaphase cells are seen at 3 hr and telophases at 4 hr (Fig. 1). The time intervals from 100 per cent labeled prophases to 100 per cent labeled meta-anaphases and telophases are 60 and 100 min, respectively. Since the duration of prophase is approximately 60 min (the time interval between the first labeled prophases and the first labeled meta-anaphases) and since about one half of all mitotic cells (of a total of 12,471 scored) are in prophase, the duration of the mitotic period in this material is about 2 hr, twice as long as the duration of prophase. This value agrees well with those of earlier studies on this material [8,11], but is only half as long as that assumed by Harris and LaCour [9]. In roots immersed for IO min in solutions of 3H-uridine or 3H-guanosine, RNA labeling is seen only in the nucleus and in particular the nucleolus of interphase through mid-prophase cells, while no labeling is observed in the cytoplasm or chromosomes of mitotic cells from late prophase through early telophase (see also [15,21]). Following a 30 to 90 min incubation period, labeled RNA appears in the cytoplasm of interphase and mitotic cells; some interphase nuclei are also labeled in DNA. As in onion and Nigella root tip cells [a], RNA synthesis continues in prophase cells in which nucleoli are present; in late telophase RNA synthesis resumes at first in the nucleolar bodies. These results are in contrast to those of Davidson [6] who reported a lack of localization of labeled RNA over the nucleolus of Vicia faba root tip cells and a cessation of RN,4 synthesis in prophase cells before the disappearance of the nucleolus. If RNA is not synthesized during the meta-anaphase stage, the rate of progress of prophase cells containing labeled RNA through meta-anaphase can be estimated in a fashion similar to that done with respect to incorporation studies using 3H-thymidine (see also [18]). Fig. 2 presents the quantitative data on the frequencies of labeled prophase and meta-anaphase cells as a function of time of exposure to 3H-uridine or 3H-guanosine. It can be seen in this figure that while about 80 per cent of prophase cells are labeled within 10 min, no labeling is seen in meta-anaphase cells. By increasing the time of immersion of roots in precursor solutions from IO to 30 min all prophases and only about 40 per cent of meta-anaphases are labeled. All of the metaanaphase cells are labeled only after about 80 min exposure to the labeled precursors.. The labeled RNA in meta-anaphase cells is located primarily in the cytoplasm. The Experimental
Cell Research 43
180
N. Ii. Lkrs et cd.
time interval between 100 per cent labeled prophases and 100 per cent labeled metaanaphases is about 50 min, a value similar to that obtained from labeling cells with 3H-thymidine (compare Figs. 1 and 2). The close agreement obtained on the rate of progress of cells from prophase through meta-anaphase by labeling cells with 3H-thymidine and 3H-uridine or 3H-guanosine
0 20 40 60 80 IGO TIME
IHR I AFTER
H3-THYMIDINE
Fig. 1.
LABELING
TiME AND
,M,N I IN H3-URIDINE IN H3-GUANOSINE
lo
I- A) Al
Fig. 2.
Fig. l.PFrequencies of labeled mitotic cells as a function of time following an initial 30 min labeling with 3H-thymidine (1 PC/ml). Data are combined from 2 experiments. Average number of mitotic cells counted at random per root: 216 prophases, 95 meta-anaphases, and 104 telophases. No. of roots at each point: 2 to 6 except 1 at 64 and at 7: hr. The autoradiographs of Feulgenstained squash preparations were exposed for l-3 weeks. Fig. 2.-Frequencies of labeled prophase and meta-anaphase cells as a function of time of exposure to 3H-uridine (50 PC/ml) and to 3H-guanosine (50 ,uc/ml). Cells were counted in sections (2 ,u) and in squash preparations (following pectinase digestion) of roots exposed to 3H-uridine, while cells were counted only in sections (3 p) of roots exposed to 3H-guanosine. Average No. of mitotic cells counted per root: 3H-uridine, 60 prophases and 52 meta-anaphases; 3H-guanosine, 36 prophases and 20 meta-anaphases. No. of roots at each point: 2 to 3. Autoradiographs were stained with Harris’ hematoxylin through the processed emulsion and exposed for 4-21 days, depending upon the duration of isotope treatment, in order to obtain heavy grain densities.
strongly suggests that the labeled cytoplasmic RNA of meta-anaphase cells was produced during prophase or earlier stages, and was transferred into the cytoplasm while the prophase cells moved to meta-anaphase. This conclusion is further borne out by grain counts over prophase and meta-anaphase cells from roots exposed to 3H-uridine for 45 and 90 min. The average number of grains over sections of prophase cells from the 45 min sample and over sections of meta-anaphase cells from the 90 min sample were 25.6 26.2 (n -45) and 21.5 +0.2 (n =45), respectively. The similarity in the grain counts over prophase and meta-anaphase cells from these two samples excludes the possibility of RNA synthesis in the meta-anaphase cell itself; most of the labeled RNA detected at meta-anaphase in the 90 min sample was produced while these cells were in prophase at least 45 min earlier. Since the average number of grains over 45 prophase cells from the 90 min sample is 68.0 +4.4, the lack of incorporation of 3H-uridine in meta-anaphase cells is not due to exhaustion of supplied precursor. While our study was in progress, Davidson [6] published his results on RNA synthesis in root meristems of Vicia faba. Although he pointed out that the synthesis Experimental
Cell Research 40
hbeled
R.XA4 in cytoplmm
of mitotic
cells
181
of RNA in prophase or interphase nuclei and subsequent transfer of this RNA into the cytoplasm accounts for the presence of labeled RNA in the cytoplasm of metaphase cells, his data on the nuclear to cytoplasmic grain ratios do not exclude the possibility of some cytoplasmic RNA synthesis in the metaphase cell itself. The present data, however, do seem to rule out this possibility. In mammalian tissue culture, metaphase cells which are arrested for some time with colchicine treatment also fail to incorporate labeled RNA precursors [15]. Furthermore, in all materials so far studied no RNA labeling is detected in metaphase and anaphase cells during the first and second meiotic divisions even after incubation of cells with labeled RNA precursors for several hours 13, 3, 5, 10, i2, 131. The results reported here lend further support to the view that mitotic (as well as meiotic) chromosome condensation renders the whole cell inactive with respect to RNA synthesis.
REFERENCES 1. APELGOT, S. and EKERT, B., J. Chim. Phys. 60, 505 (1963). 2. DAS, N. K., Science 140, 1231 (1963). 3. __ Expfl Cell Res. In press. 1. DAS, N. K., LUYKX, P. and ALFERT, M., Deuelop. Biol. In press. 5. DAS, N. K., SIEGEL, E. P. and ALFERT, M., J. dell Biol. 25,387 (1965). 6. DAVIIISOX, D., Bxptl Cell Res. 35, 317 (1964). 7. EVASS, H. J., NE.&, G. .J. and TOSKINSON, S. M., J. Genetics 55, 487 (1957). X. EVANS, H. J. and SCOTT, D., Genetics 49, 17 (1964). 9. HARRIS, H. and LACOUR, L. F., Sature 200, 227 (1963). IO. HENDEHSOS, S. A., Chromosoma 15, 345 (1964). 11. HOWARD, A. and PELC, S. R., Heredity 6 (suppl.), 261 (1952). 12. MOSESI, V., J. Cell Biof. 22, 521 (1964). 13. MUCICENTIIALER, F. A., Expff Cell Res. 35, 531 (1964). 14. SICOS, V. and GELLOT, S., Ezptl Cell Res. 33, 29 (1964). 15. PRESCOTT, D. M., in J. S. DAVIDSON and W. E. COHX (eds.), Progress in Nucleic Acid Research and Molecular Biology, vol. 3, p. 33. Academic Press, New York, 1964. 16. PRESCOTT, D. M. and BENDER, M. A., Ezptf Cell Res. 26, 260 (1962). 15. TAKATS, S. T. and S~XELLIE, R. M. S., J. Cell Riol. 17, 59 (1963). 18. TAYLOR, J. H., Ann. S.Y. ilcad. Sci. 90, 409 (1960). 19. __ J. Hiophys. Biochem. Cyfof. 7, 455 (1960). 20. WIMBER, D. E., Am. J. Hofan. 47, 828 (1960). 21. WOODS, P. S., in .J. S. MITCIIELL (cd.), The Cell Nucleus, p. 127. Academic Press, New York, 1960.
Esperimenfal
Cell Research 40
ORIGIN MITOTIC N. K. DAS,
Department
OF LABELED ROOT
TIP
ELSIE
of Zoology,
RNA CELLS
IN THE OF WCIA
P. SIEGEL
University
CYTOPLASM
OF
FABA
and M. ALFERT
of California,
Berkeley,
C&j’.,
U.S.A.
Received May 24, 1965
PULSE-LABELING experiments have shown that meta-anaphase cells of both plant and animal tissues are inactive with respect to RNA synthesis [2, 16, 181. Harris and LaCour [9], however, pointed out that pulse-labeling experiments may not be sensitive enough to detect low levels of cytoplasmic RNA synthesis in mitotic cells as well as in interphase cells. They observed the presence of labeled RNA in the cytoplasm of meta-anaphase cells of Vicia faba root tip meristems exposed to tritiated guanosine for 30 to 90 min. The duration of mitosis in this tissue had been assumed to be about 4 hr [7]. Since prophase occupies about one half of the total mitotic period and since RNA is not produced by the condensed meta-anaphase chromosomes they postulate that the observed cytoplasmic labeling is evidence of RNA synthesis in the cytoplasm during mitosis. In earlier [ll] and more recent [8] studies, however, the duration of mitosis in Vicia faba root meristem cells has been found to be about 2 rather than 4 hr. It seems very likely, therefore, that a considerable number of prophase cells, which are active in RNA synthesis, might have moved to meta-anaphase within 30 min, the earliest incorporation time used by Harris and LaCour. This possibility is tested in the present autoradiographic study. The gradual progression of cells to meta-anaphase has been followed by labeling with tritiated thymidine (see [19, 201) and the resulting data are compared with those obtained from the incorporation of tritium labeled RNA precursors. In experiments designed to determine the duration of the mitotic period, secondary roots of Vicia faba grown in aerated tap water were exposed to 1 +/ml of 3H-thymidine (spec. act. 3 and 6.7 c/miM’) for 30 min, washed thoroughly, and allowed to grow further in aerated tap water. Samples were collected from 0 to 10: hr. In other experiments, secondary roots were immersed in tap water with added 50 /+/ml of 3H-uridine (spec. act. 3.7 c/m&Z) or 50 ,+/ml of 3H-guanosine (spec. act. 1.5 cimM) for 10 to 90 min. All samples were fixed in acetic alcohol. Roots treated with 3Hthymidine were stained with Feulgen and squashed on slides. Since the hydrolysis in 1 N HCl used in the Feulgen staining procedure removes RNA, roots exposed to 3H-uridine or 3H-guanosine were softened by hydrolyzing in 10 per cent pectinase solution at pH 4 for 1 hr prior to squashing, or 2- 3 /A paraffin sections were prepared; these slides were then treated with cold TCA to remove acid soluble nucleotides. Autoradiographs were prepared by dipping slides in liquid emulsion (NTB2). Cells containing labeled RNA were stained with Harris’ hematoxylin through the processed emulsion. Some of the slides were digested with RNase, DNase, or hot TCA to remove This study was supported by grants from the National U.S. Public Health Service (RG-6025). Experimental
Cell Research 40
Science Foundation
(GB-1642)
and the
Ltrbeled RLYL4in cytoplasm of mitotic
cells
nucleic acids prior to coating slides with emulsion. A relatively long exposure of emulsion resulted in detection of some radioactivity, especially in the cytoplasm, that \yas not removable by nuclease or hot TCA digestion, suggesting that tritium is also incorporated into compounds other than nucleic acids (see also 15, 14, 171). Such non-specific labeling probably results from the small amount of impurities that is always present in commercially available labeled nucleosides [l]. Autoradiographic exposures (1-21 days) were chosen so that grains were due predominantly to the tritium incorporated into nucleic acids. The frequency of mitotic cells labeled 3H-uridine, or 3H-guanosine was determined from squashes with 3H-thymidinc, as well as from sections. A preponderance of labeled cells had grain densities many times greater than background. Cells were considered to be labeled, however, if the number of grains exceeded five above background. Grain counts (corrected for background) were also made over sectioned mitotic cells labeled with 3H-uridine. At about 2 hr after an initial 30-min exposure of roots to 3H-thymidine the first labeled prophases begin to appear and 4 hr later all prophases are labeled (Fig. 1). The first labeled meta-anaphase cells are seen at 3 hr and telophases at 4 hr (Fig. 1). The time intervals from 100 per cent labeled prophases to 100 per cent labeled meta-anaphases and telophases are 60 and 100 min, respectively. Since the duration of prophase is approximately 60 min (the time interval between the first labeled prophases and the first labeled meta-anaphases) and since about one half of all mitotic cells (of a total of 12,471 scored) are in prophase, the duration of the mitotic period in this material is about 2 hr, twice as long as the duration of prophase. This value agrees well with those of earlier studies on this material [8,11], but is only half as long as that assumed by Harris and LaCour [9]. In roots immersed for IO min in solutions of 3H-uridine or 3H-guanosine, RNA labeling is seen only in the nucleus and in particular the nucleolus of interphase through mid-prophase cells, while no labeling is observed in the cytoplasm or chromosomes of mitotic cells from late prophase through early telophase (see also [15,21]). Following a 30 to 90 min incubation period, labeled RNA appears in the cytoplasm of interphase and mitotic cells; some interphase nuclei are also labeled in DNA. As in onion and Nigella root tip cells [a], RNA synthesis continues in prophase cells in which nucleoli are present; in late telophase RNA synthesis resumes at first in the nucleolar bodies. These results are in contrast to those of Davidson [6] who reported a lack of localization of labeled RNA over the nucleolus of Vicia faba root tip cells and a cessation of RN,4 synthesis in prophase cells before the disappearance of the nucleolus. If RNA is not synthesized during the meta-anaphase stage, the rate of progress of prophase cells containing labeled RNA through meta-anaphase can be estimated in a fashion similar to that done with respect to incorporation studies using 3H-thymidine (see also [18]). Fig. 2 presents the quantitative data on the frequencies of labeled prophase and meta-anaphase cells as a function of time of exposure to 3H-uridine or 3H-guanosine. It can be seen in this figure that while about 80 per cent of prophase cells are labeled within 10 min, no labeling is seen in meta-anaphase cells. By increasing the time of immersion of roots in precursor solutions from IO to 30 min all prophases and only about 40 per cent of meta-anaphases are labeled. All of the metaanaphase cells are labeled only after about 80 min exposure to the labeled precursors.. The labeled RNA in meta-anaphase cells is located primarily in the cytoplasm. The Experimental
Cell Research 43
180
N. Ii. Lkrs et cd.
time interval between 100 per cent labeled prophases and 100 per cent labeled metaanaphases is about 50 min, a value similar to that obtained from labeling cells with 3H-thymidine (compare Figs. 1 and 2). The close agreement obtained on the rate of progress of cells from prophase through meta-anaphase by labeling cells with 3H-thymidine and 3H-uridine or 3H-guanosine
0 20 40 60 80 IGO TIME
IHR I AFTER
H3-THYMIDINE
Fig. 1.
LABELING
TiME AND
,M,N I IN H3-URIDINE IN H3-GUANOSINE
lo
I- A) Al
Fig. 2.
Fig. l.PFrequencies of labeled mitotic cells as a function of time following an initial 30 min labeling with 3H-thymidine (1 PC/ml). Data are combined from 2 experiments. Average number of mitotic cells counted at random per root: 216 prophases, 95 meta-anaphases, and 104 telophases. No. of roots at each point: 2 to 6 except 1 at 64 and at 7: hr. The autoradiographs of Feulgenstained squash preparations were exposed for l-3 weeks. Fig. 2.-Frequencies of labeled prophase and meta-anaphase cells as a function of time of exposure to 3H-uridine (50 PC/ml) and to 3H-guanosine (50 ,uc/ml). Cells were counted in sections (2 ,u) and in squash preparations (following pectinase digestion) of roots exposed to 3H-uridine, while cells were counted only in sections (3 p) of roots exposed to 3H-guanosine. Average No. of mitotic cells counted per root: 3H-uridine, 60 prophases and 52 meta-anaphases; 3H-guanosine, 36 prophases and 20 meta-anaphases. No. of roots at each point: 2 to 3. Autoradiographs were stained with Harris’ hematoxylin through the processed emulsion and exposed for 4-21 days, depending upon the duration of isotope treatment, in order to obtain heavy grain densities.
strongly suggests that the labeled cytoplasmic RNA of meta-anaphase cells was produced during prophase or earlier stages, and was transferred into the cytoplasm while the prophase cells moved to meta-anaphase. This conclusion is further borne out by grain counts over prophase and meta-anaphase cells from roots exposed to 3H-uridine for 45 and 90 min. The average number of grains over sections of prophase cells from the 45 min sample and over sections of meta-anaphase cells from the 90 min sample were 25.6 26.2 (n -45) and 21.5 +0.2 (n =45), respectively. The similarity in the grain counts over prophase and meta-anaphase cells from these two samples excludes the possibility of RNA synthesis in the meta-anaphase cell itself; most of the labeled RNA detected at meta-anaphase in the 90 min sample was produced while these cells were in prophase at least 45 min earlier. Since the average number of grains over 45 prophase cells from the 90 min sample is 68.0 +4.4, the lack of incorporation of 3H-uridine in meta-anaphase cells is not due to exhaustion of supplied precursor. While our study was in progress, Davidson [6] published his results on RNA synthesis in root meristems of Vicia faba. Although he pointed out that the synthesis Experimental
Cell Research 40
hbeled
R.XA4 in cytoplmm
of mitotic
cells
181
of RNA in prophase or interphase nuclei and subsequent transfer of this RNA into the cytoplasm accounts for the presence of labeled RNA in the cytoplasm of metaphase cells, his data on the nuclear to cytoplasmic grain ratios do not exclude the possibility of some cytoplasmic RNA synthesis in the metaphase cell itself. The present data, however, do seem to rule out this possibility. In mammalian tissue culture, metaphase cells which are arrested for some time with colchicine treatment also fail to incorporate labeled RNA precursors [15]. Furthermore, in all materials so far studied no RNA labeling is detected in metaphase and anaphase cells during the first and second meiotic divisions even after incubation of cells with labeled RNA precursors for several hours 13, 3, 5, 10, i2, 131. The results reported here lend further support to the view that mitotic (as well as meiotic) chromosome condensation renders the whole cell inactive with respect to RNA synthesis.
REFERENCES 1. APELGOT, S. and EKERT, B., J. Chim. Phys. 60, 505 (1963). 2. DAS, N. K., Science 140, 1231 (1963). 3. __ Expfl Cell Res. In press. 1. DAS, N. K., LUYKX, P. and ALFERT, M., Deuelop. Biol. In press. 5. DAS, N. K., SIEGEL, E. P. and ALFERT, M., J. dell Biol. 25,387 (1965). 6. DAVIIISOX, D., Bxptl Cell Res. 35, 317 (1964). 7. EVASS, H. J., NE.&, G. .J. and TOSKINSON, S. M., J. Genetics 55, 487 (1957). X. EVANS, H. J. and SCOTT, D., Genetics 49, 17 (1964). 9. HARRIS, H. and LACOUR, L. F., Sature 200, 227 (1963). IO. HENDEHSOS, S. A., Chromosoma 15, 345 (1964). 11. HOWARD, A. and PELC, S. R., Heredity 6 (suppl.), 261 (1952). 12. MOSESI, V., J. Cell Biof. 22, 521 (1964). 13. MUCICENTIIALER, F. A., Expff Cell Res. 35, 531 (1964). 14. SICOS, V. and GELLOT, S., Ezptl Cell Res. 33, 29 (1964). 15. PRESCOTT, D. M., in J. S. DAVIDSON and W. E. COHX (eds.), Progress in Nucleic Acid Research and Molecular Biology, vol. 3, p. 33. Academic Press, New York, 1964. 16. PRESCOTT, D. M. and BENDER, M. A., Ezptf Cell Res. 26, 260 (1962). 15. TAKATS, S. T. and S~XELLIE, R. M. S., J. Cell Riol. 17, 59 (1963). 18. TAYLOR, J. H., Ann. S.Y. ilcad. Sci. 90, 409 (1960). 19. __ J. Hiophys. Biochem. Cyfof. 7, 455 (1960). 20. WIMBER, D. E., Am. J. Hofan. 47, 828 (1960). 21. WOODS, P. S., in .J. S. MITCIIELL (cd.), The Cell Nucleus, p. 127. Academic Press, New York, 1960.
Esperimenfal
Cell Research 40