Synthesis of Full Length cDNAs from Four Partially Purified Oviduct mRNAs*

Total poly(A)-containing RNA prepared from hen oviduct and centrifuged on an isokinetic sucrose gradient displays four peaks of optical absorbance. These have been identified by translation in vitro as lysozyme, ovomucoid, ovalbumin, and conalbumin mRNAs. Isolation and recentrifugation of the peaks results in partial purification of each mRNA. Molecular weights have been determined for the mRNAs on agarose gels containing 20 mM methylmercury hydroxide. Each mRNA possesses a number of apparently untranslated nucleotides ranging from approximately 900 bases for ovalbumin and conalbumin mRNAs to 200 bases for ovomucoid and lysozyme mRNAs. The mRNAs have been copied with avian myeloblastosis virus reverse transcriptase. Each with the exception of conalbumin gives rise to a high proportion of full length cl)NA. Several parameters previously reported to influence the size distribution of cI)NA had no effect on the length of cl)NA made from any mRNA fraction. The proportion of full length depend on the reverse transcriptase immature results in oviduct

Filters were washed with 20 to 30 ml of cold 5% trichloroacetic acid containing 0.5% sodium pyrophosphate, placed in scintillation vials, and 10 ml of scintillation mixture added. In experiments using "H, filters were soaked in 1 ml of 0.1 N NaOH for about 30 min at 50" before being added to scintillation mixture.

RESULTS
The fully estrogen-stimulated hen oviduct is highly specialized. Most of its protein synthesis is devoted to the four eggwhite proteins, ovalbumin, conalbumin, ovomucoid, and lysozyme which comprise approximately 64%, 12%, 8.5%, and 1.5% of soluble protein synthesis, respectively (3). Since these four proteins differ substantially in size from one another (see Table II), we anticipated that centrifugation of oviduct total poly(A)-containing RNA through sucrose gradients would resolve the four mRNAs. Poly(A)-containing RNA was sedimented through an isokinetic 5 to 20% sucrose gradient and the AzGO pattern monitored. The resulting profile, Fig. la, shows four discrete peaks with the relative abundances and migrations expected for the four mRNAs.
The mRNA activities were identified by translating each gradient fraction in vitro and immunoprecipitating the various products separately. The gradient shown in Fig. 1 was collected into 18 fractions. Each was ethanol-precipitated, resuspended in water, and an equal aliquot used to program a rabbit reticulocyte lysate translation system as described under "Experimental Procedures. " :'H-labeled reaction products were precipitated with either anti-conalbumin, anti-ovalbumin, or anti-ovomucoid (insufficient anti-lysozyme was available for this experiment).
As shown in Fig. lb, the mRNA translation activities for conalbumin, ovalbumin, and ovomucoid are coincident with the appropriate absorbance peak.
Two preparations of enriched mRNA fractions were made. Total poly(A)-containing RNA was sedimented as described under "Experimental Procedures" and the peak fractions for each activity pooled. After concentration by ethanol precipitation, pooled fractions were recentrifuged on identical gradients either three times (Experiment 1) or once (Experiment 2). While the additional purification in Experiment 1 simplified subsequent mRNA characterization, a single recentrifugation resulted in mRNAs enriched sufficiently for analyzing the synthesis of double-stranded cDNA. Repeated centrifugation was particularly useful for separating ovalbumin and conalbumin mRNAs which sometimes migrated as faster sedimenting aggregates (see "Experimental Procedures"). Partially purified mRNAs from the two experiments were on an isokinetic 5 to 20% sucrose gradient prepared in SET. The sample was heated in SET for 10 min at 68" before centrifugation.
Centrifugation was for 7 h at 20" and 41,000 rpm in a Beckman SW 41 rotor. The gradient was collected into 18 fractions which were ethanol-precipitated, washed with 70% ethanol, dried, and resuspended in 100 ~1 of water. partial purification for the expected species and substantial separation from the other three (Table I). Table Ia presents the specific activity of each mRNA in total poly(A)-containing RNA and in the final enriched fraction and the derived fold purification. Table Ib presents the results of a cross-translation experiment in which the activities of the assayable contaminants were determined. Our interpretation of these specific activities in terms of per cent contamination is also shown. Three conclusions can be drawn from these data. First the previously unidentified smallest peak in Fig. 1 co-migrates with lysozyme mRNA, since that purified fraction is enriched 17-fold for lysozyme translational activity in vitro. The nearest neighboring mRNA fraction, ovomucoid, shows little lysozyme translational activity. Second, each peak displays an increased specific activity for the protein's translation in vitro with a fold enrichment consistent with its rate of synthesis in ho. Finally, purification of each peak results in a large decrease in the activity of other specific translatable mRNAs. Expressed as per cent contamination (see legend to Table I) it appears that no fraction is contaminated by any of the other three more than 10%.
The mRNA fractions from Experiment 2 were assessed for purity by translation in the mRNA-dependent reticulocyte lysate and gel electrophoresis in formamide. The reticulocyte lysate was rendered mRNA-dependent by hydrolysis of endogenous mRNA with calcium-dependent micrococcal nuclease which was subsequently inactivated by the addition of EGTA (18, see "Experimental Procedures"). This treatment reduced   Fig. 6a). No prominent band of ovomucoid or lysozyme mRNA can be distinguished even though these mRNAs co-migrate with major peaks of optical absorbance in total poly(A)-containing RNA centrifuged on sucrose gradients (Fig. 1). The inability to visualize discrete bands of ovomucoid or lysozyme mRNAs in the electrophoretic profile of total oviduct mRNA can be attributed to at least two possible factors: proportionally greater broadening of bands by poly(A) on small mRNAs and the presence of small RNA fragments which remain intact during centrifugation in SET. The electrophoretic profile of cDNA synthesized from total poly(A)-containing RNA (Fig. 6b) is consistent with these interpretations and suggests that not all RNAs in Fig. 6u  Truth 1 and 2 of Fig. 6a, no full length conalbumin cDNA is visible in the autoradiogram (Fig. 6b). Conalbumin mRNA must therefore be a poor template for synthesis of full length cDNA relative to the other three mRNAs. As shown in Figs. 10 and 12, only a small proportion of the cDNA made from the partially purified conalbumin mRNA fraction is full length.
Parameters Affecting Synthesis of cDNA maximal yield of full length ovalbumin cDNA (39). When we examined the time course of ovalbumin cDNA synthesis at 37", 42", and 46" little effect of temperature on the total incorporation after 1 h was observed (Fig. 7). The reaction was rapid and increasing the incubation temperature from 37-46 accelerated the initial rate. However, the length distribution of fully denatured ovalbumin cDNA made at the three temperatures was indistinguishable for l-h incubations (Fig. 8   migration on gels which were sliced and counted (data not shown). Effect of KC1 Concentration-Another possible parameter affecting the synthesis of full length cDNA is KC1 concentration. Synthesis in the absence of salt has been reported to increase the proportion of full length product using ovalbumin mRNA as template with AMvirus reverse transcriptase (39). We used each of the four mRNA fractions from Experiment 2 as templates for reverse transcriptase in reactions containing KC1 at concentrations between 0 and 140 mM. With each template maximal incorporation was observed at 70 mM KC1 (Fig. 9). The similar contour of the curves suggests that the effect of KC1 concentration on cDNA yield is not templatespecific. Poor incorporation into conalbumin cDNA correlates with the absence of full length conalbumin cDNA in Fig. 6b.
The length distributions of cDNAs synthesized with varying concentrations of KC1 were determined on denaturing agarose gels. In the case of ovalbumin cDNA (Fig. 1Oo) the size distribution of each of these products is very similar. Each is composed of a major band migrating in the position expected for full length ovalbumin cDNA and a similar distribution of partial products. The same result was obtained with cDNA synthesized in 0 or 140 mM KC1 using the other three mRNA fractions (Fig. 10, b to cl). We conclude that while KC1 concentration does have an effect on the yield of cDNA, it has little influence on the product size distribution.
Effect RNA was electrophoresed on barbital-buffered formamide, 3.5% acrylamide gels as described under "Experimental Procedures." Gel 1 was loaded with 108 pg and Gel 2 with 42 p,g plus 2 pg of E. coli rRNA.
Both gels contain 2 pg of E. coli tRNA. The positions of 23 S and 16 S rRNAs on Gel 2 were determined by electrophoresis of these markers on a separate gel run in parallel (data not shown).
The bands of ovalbumin (0~) and conalbumin (Con) mRNAs and the approximate positions of ovomucoid (Mu) and lysozyme (Lys) mRNAs were determined from their migrations relative to E. coli rRNA. Gels were stained and viewed as described under "Experimental Procedures." In b, [32PlcDNA was synthesized from total poly(A)-containing RNA in a standard reaction using [32P1dGTP (16 Cilmmol), electrophoresed on a 20 mM CH,HgOH-2% agarose slab gel and autoradiographed as described under "Experimental Procedures." DNA markers (not shown) were the nicktranslated Hind111 fragments of SV40 DNA. The arrow marks the position expected for full length conalbumin cDNA.
High concentrations of deoxynucleotide triphosphates have been reported to increase the average size of cDNA (39-43). In two of these reports full length cDNA could be obtained with low dXTP concentrations and the proportion of long cDNA increased by raising dXTP concentration (40,41). In the other reports (39, 40,43) only a small amount of the cDNA was long or full length at all dXTP concentrations.
Where discussed (39, 41) investigators have found higher dXTP concentrations to enhance the yield of cDNA. We also observed this effect on total incorporation for reactions between 50 and 100 PM dXTPs with the four mRNA templates (Fig. 11). However, within the range of dXTP concentrations, no change in the size distribution of cDNA could be detected for any of the mRNA fractions (Fig. 12). Ovalbumin cDNA in each track of Fig. 12~ was synthesized with a different concentration of dXTP present, using a constant specific radioactivity. cDNAs were synthesized from the other mRNA fractions (Fig. 12,  and electrophoresed on a 20 mM CH,HgOH-1.5% agarose slab gel. Track 2 is an aliquot of the same reaction after a 5-min reaction time. Tracks 4 to 6 are ovalbumin cDNA synthesized in the same manner as ovalbumin cDNA seen in Track 3 except for differences in reaction temperature: Track 4 at 37", Track 5 at 42", and Track 6 at 46". An equal amount of radioactivity was applied to Tracks 2 to 6. Truck I is the nick-translated Hind111 fragments of SV40 DNA, overexposed in order to obtain the desired exposures for other tracks.

Comparison of Enzyme Preparations
-All cDNA shown in Figs. 7 to 12 was prepared with AMvirus reverse transcriptase, Lot G-1176 at 10 unitslpg of RNA. We also tested a second enzyme preparation, Lot G-577 from the same source for its ability to catalyze the synthesis of full length ovalbumin cDNA. The relative yields and size distributions of ovalbumin cDNA made with varying amounts of the two enzyme lots is presented in Fig. 13. Up to 7 unitslpg of mRNA, yields with the two lots are comparable. At enzyme:RNA ratios above 7 pholia t3H]rRNA (40,000 cpm/pg) to 50 ~1 of reverse transcriptase reaction mixtures containing either 3.5 or 60 units of one of the two enzyme preparations and incubating for 60 min at 42". Following incubation, 1 ~1 was acid-precipitated and the remainder of each sample concentrated by ethanol precipitation and electrophoresed on 98% formamide, 3.2% acrylamide tube gels (23). After electrophoresis gels were sliced and counted (see "Experimental Procedures"). Although each sample contained the same amount of acid-precipitable radioactiv- -In order to determine whether the major species of cDNA synthesized from each of the mRNAs was the full length copy, we determined their molecular weights on Hind111 fragments of SV40 DNA were run in adjacent slots and gave calibration curves in which migration was linear with the log molecular weight over the desired range. Table III gives the average of several molecular weight   determinations for the major band in each cDNA. These lengths agree well with the expected values if cDNA synthesis is initiated near the 5' end of mRNA poly(A) tracts.
The Sl nuclease resistance of each cDNA was determined for the reverse transcriptase product before and after its denaturation at 100" for 5 min in 20 mM NaCl (Table III). As previously observed for globin cDNA (411, the high Sl nuclease resistance (80 to 85%) of the native structure suggests that most cDNA remains bound to template after synthesis. The Sl nuclease resistance of denatured cDNA was determined at two NaCl concentrations, 300 and 50 mM. The Sl resistance after boiling dropped to less than lo%, consistent with the cDNA having been freed from template. DISCUSSION We have obtained conalbumin, ovalbumin, ovomucoid, and lysozyme mRNAs in a rapid, convenient fashion and found them to be suitable templates for reverse transcriptase.
Although immunoprecipitation of specific polysomes (11) would probably yield mRNAs of greater purity, the predominance of these mRNAs in the oviduct and their differences in sedimentation rates have allowed us to obtain sufficient enrichments by size fractionation of total poly(A)-containing RNA on sucrose gradients (Fig. 1).
All four mRNAs are longer than necessary to code for the polypeptides (Table II). In the case of ovalbumin and conalbumin mRNAs there appear to be approximately 900 untranslated nucleotides. Inasmuch as the protein products synthesized in vitro are not noticeably greater in size than the authentic proteins, we cannot ascribe many extra nucleotides to large precursors, although a signal peptide (44) of approximately 20 to 30 amino acids might be undetected. Precursor sequences of about 20 amino acids were recently described for ovomucoid and lysozyme (35) and are taken into account in our estimate of untranslated nucleotides for the respective mRNAs. In each case some of the nucleotides, probably about 50, can be accounted for by a 3'-polyadenylic acid sequence. Estimates of the molecular weight of ovalbumin mRNA have varied considerably (11,38,45). The difficulties in determining mRNA molecular weights by comparison to rRNA standards of defined size stem from differences in base composition and secondary structure not readily eliminated even in 98% formamide (37). The methylmercury concentration dependence of electrophoretic mobility (Fig. 4 and Ref. 21) is consistent with all RNAs tested having been fully denatured by 20 mM CH,HgOH. Calculations of mRNA molecular weight from relative mobilities in this medium therefore appear to be valid.
Conalbumin mRNA is unusual in at least two respects. First, it is translated poorly in the rabbit reticulocyte lysate, whether using the intact system (12) or that rendered mRNA dependent by prior treatment with micrococcal nuclease. Second, conalbumin mRNA is a poor template for full length cDNA synthesis (Fig. 6). Data on total incorporation suggest that conalbumin mRNA is a relatively inefficient template for reverse transcriptase although uncertainty about the purity of the conalbumin mRNA fraction in Experiment 2 (Fig. 2) does not allow firm conclusions. One of the assumptions in defining abundance classes has been that all mRNAs have equal transcriptional efficiencies with reverse transcriptase (46,47). This assumption may not be valid for oviduct mRNAs.
In investigating several of the parameters involved in cDNA synthesis we found the total incorporation to be influenced by the time of incubation,