Current and future practices in surgical retraction.

Retraction of tissues and anatomical structures is an essential component of all forms of surgery. The means by which operative access is gained through retraction are many and diverse. In this article, the various forms of retraction methods currently available are reviewed, with special reference to hand held, self-retaining and compliant techniques. The special challenges posed by laparoscopic surgery are considered and future developments in new retraction techniques are anticipated.

Analysis of codon:anticodon interactions within the ribosome provides new insights into codon reading and the genetic code structure.

Although the decoding rules have been largely elucidated, the physical-chemical reasons for the "correctness" of codon:anticodon duplexes have never been clear. In this work, on the basis of the available data, we propose that the correct codon:anticodon duplexes are those whose formation and interaction with the ribosomal decoding center are not accompanied by uncompensated losses of hydrogen and ionic bonds. Other factors such as proofreading, base-base stacking and aminoacyl-tRNA concentration contribute to the efficiency and accuracy of aminoacyl-tRNA selection, and certainly these factors are important; but we suggest that analyses of hydrogen and ionic bonding alone provides a robust first-order approximation of decoding accuracy. Thus our model can simplify predictions about decoding accuracy and error. The model can be refined with data, but is already powerful enough to explain all of the available data on decoding accuracy. Here we predict which duplexes should be considered correct, which duplexes are responsible for virtually all misreading, and we suggest an evolutionary scheme that gave rise to the mixed boxes of the genetic code.

An allosteric synthetic DNA.

Allosteric DNA oligonucleotides are potentially useful diagnostic reagents. Here we develop a model system for the study of allosteric interactions in DNAs. A DNA that binds either Cibacron blue or cholic acid was isolated and partially characterized. Isolation was performed using a multi-stage SELEX. First, short oligos that bind either Cibacron blue or cholic acid were enriched from random oligonucleotide pools. Then, members of the two pools were fused to form longer oligos, which were then selected for theability to bind Cibacron blue columns and elute with cholic acid. One resulting isolate (A22) was studied. Dye- and cholate-binding functions can be separated on sequences from the 5'- and 3'-regions, respectively. Ligand-column affinity assays indicate that each domain binds only its respective ligand. However, the full-length A22 will bind either dye or cholate columns and elute with the other ligand, as if binding by the ligands is mutually exclusive. Furthermore, S1 nuclease protection assays show that Cibacron blue causes a structural change in A22 and that cholic acid inhibits this change. This system will be useful for elucidating mechanisms of allosteric interactions in synthetic DNAs.

tRNA(2Gln) mutants that translate the CGA arginine codon as glutamine in Escherichia coli.

We present a novel missense suppression system for the selection of tRNA(2GIn) mutants that can efficiently translate the CGA (arginine) codon as glutamine. tRNA(2Gln) mutants were cloned from a partially randomized synthetic gene pool using a plasmid vector that simultaneously expresses the tRNA gene and, to ensure efficient aminoacylation, the glutamine aminoacyl-tRNA synthetase gene (glnS). tRNA mutants that insert glutamine at CGA were selected as missense suppressors of a lacZ mutant (lacZ625(CGA)) that contains CGA substituted for an essential glutamine codon. Preliminary characterizations of four suppressors is presented. All of them contain two anticodon mutations: C-->U at position 34 and U-->C at position 35, which allow for cognate translation of CGA. U35 was previously shown to be an important determinant for glutaminylation of tRNA(2Gln) in vitro; suppression in vivo requires overexpression of the glutaminyl-tRNA synthetase gene (glnS). One tRNA variant contains no further mutations and has the highest missense suppression activity (8%). Three other isolates each contain an additional point mutation that alters suppression efficiency. This system will be useful for further studies of tRNA structure and function. In addition, because relatively efficient translation of the rare CGA codon as glutamine is not toxic for Escherichia coli, it may be possible to translate this sense codon with other alternate meanings, a property which could greatly facilitate protein engineering.

The methyl group of the N6-methyl-N6-threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA.

tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3' of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA(Thr1)GGU and tRNA(Thr3)GGU) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNA(Thr)GGU species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-L-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA(Thr)GGU to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNA(Thr)GGU species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNA(Thr)GGU slightly reduces the efficiency of this tRNA to read cognate codon ACC.

Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner.

In Salmonella typhimurium seven tRNA species specific for leucine, proline and arginine have 1-methylguanosine (m1G) next to and 3' of the anticodon (position 37 of tRNA), five tRNA species specific for phenylalanine, serine, tyrosine, cysteine and tryptophan have 2-methylthio-N-6-(cis-hydroxy)isopentenyladenosine (ms2io6A) in the same position of the tRNA, and four tRNA species, specific for leucine and proline, have pseudouridine (Psi) as the last 3' nucleotide in the anticodon loop (position 38) or in the anticodon stem (positions 39 and 40). Mutants deficient in the synthesis of these modified nucleosides have been used to study their role in the first step of translation elongation, i.e. the aa-tRNA selection step in which the ternary complex (EF-Tu-GTP-aa-tRNA) binds at the cognate codon in the A-site on the mRNA programmed ribosome. We have found that the Psi present in the anticodon loop (position 38) stimulates the selection of tRNA specific for leucine whereas Psi in the anticodon stem did not affect the selection of tRNA specific for proline. The m1G37 strongly stimulates the rate of selection of the three tRNA species specific for proline and one tRNA species specific for arginine but has only minor or no effect on the selection of the three tRNA species specific for leucine. Likewise, the ms2io6A, present in the same position as m1G37 but in another subset of tRNA species, stimulates the selection of tRNA specific for tyrosine, stimulates to some extent also tRNA species specific for cysteine and tryptophan, but has no influence on the rate of selection of tRNA specific for phenylalanine. We conclude that function of m1G and ms2io6A present next to and 3' of the anticodon influences the in vivo aa-tRNA selection in a tRNA-dependent manner.

Analyses of frameshifting at UUU-pyrimidine sites.

Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts. Here, several approaches are used to analyze frameshifting at such sites. The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies. Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting. Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site. The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting. In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame. This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex. It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA. Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA. In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU. It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon. In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon. Thus, those base modifications may limit frameshifting at UUU codons. Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage.

Selection of aminoacyl-tRNAs at sense codons: the size of the tRNA variable loop determines whether the immediate 3' nucleotide to the codon has a context effect.

Codon context can affect translational efficiency by several molecular mechanisms. The base stacking interactions between a codon-anticodon complex and the neighboring nucleotide immediately 3' can facilitate translation by amber suppressors and the tRNA structure is also known to modulate the sensitivity to context. In this study the relative rates of aminoacyl-tRNA selection were measured at four sense codons (UGG, CUC, UUC and UCA), in all four 3' nucleotide contexts, through direct competition with a programmed frameshift at a site derived from the release factor 2 gene. Two codons (UGG and UUC) are read by tRNAs with small variable regions and their rates of aminoacyl-tRNA selection correlated with the potential base stacking strength of the 3' neighboring nucleotide. The other two codons (CUC and UCA) are read by tRNAs with large variable regions and the rate of selection of the aminoacyl-tRNAs in these cases varied little among the four contexts. Re-examination of published data on amber suppression also revealed an inverse correlation between context sensitivity and the size of the variable region. Collectively the data suggest that a large variable loop in a tRNA decreases the influence of the 3' context on tRNA selection, probably by strengthening tRNA-ribosomal interactions.

Decoding with the A:I wobble pair is inefficient.

tRNAs with inosine (I) in the first position read three codons ending in U, C and A. However, A-ending codons read with I are rarely used. In Escherichia coli, CGA/U/C are all read solely by tRNAICGArg. CGU and CGC are very common codons, but CGA is very rare. Three independent in vivo assays show that translation of CGA is relatively inefficient. In the first, nine tandem CGA cause a strong rho-mediated polar effect on expression of a lacZ reporter gene. The inhibition is made more extreme by a mutation in ribosomal protein S12 (rpsL), which indicates that ribosomal binding by tRNAICGArg is slow and/or unstable in the CGA cluster. The second assay, in which codons are substituted for the regulatory UGA of the RF2 frameshift, confirms that aa-tRNA selection is slow and/or unstable at CGA. In the third assay, CGA is found to be a poor 5' context for amber suppression, which suggests that an A:I base pair in the P site can interfere with translation of a codon in the A site. Two possible errors, frameshifting and premature termination by RF2, are not significant causes for inefficiency at CGA. It is concluded that the A:I pair destabilizes codon:anticodon complexes during two successive ribosomal cycles, and it is suggested that these properties contribute to the rare usage of codons read with the A:I base pair.

Evidence that GHN phase bias does not constitute a framing code.

It has been suggested that a triplet repeated pattern found in coding sequences, the G-nonG-N or GHN phase bias, serves a framing function during protein synthesis. To test this idea, the framing characteristics of a highly GHN biased sequence are examined. No effects on reading frame maintenance are observed despite the use of sensitive frameshift assays. Specifically, first the GHN phase is not more accurate than the alternative overlapping phases (i.e., HNG and NGH). Second, ribosomes do not exhibit any significant tendency to slip from the alternative frames into the GHN pahse. In addition, examination of Escherichia coli programmed frameshift sites does not support roles for GHN phase bias in programmed frameshifting. Framing functions for GHN phase bias, if they occur at all, must be extremely limited.

Analysis of effects of tRNA:message stability on frameshift frequency at the Escherichia coli RF2 programmed frameshift site.

The codon that is in-frame prior to +1 frameshifting at the E.coli prfB (RF2 gene) frameshift site is randomized to create thirty-two variants. These alleles vary 1000-fold in frameshift-dependent expression in fusions to lacZ. Frameshifting is more frequent at sites where the in-frame codon ends in uridine, as if third position wobble pairs to message uridine facilitate slippage into the +1 frame. Consistent with other studies of programmed frameshift sites, efficient frameshifting depends on stable message:tRNA base pairs after rephasing. For complexes with mispairs, frameshift frequency depends on the nature, number, and position of mispairs. Central purine:purine mispairs are especially inhibitory. Relative stabilities of +1 rephased complexes are estimated from published data on the stabilities of tRNA:tRNA complexes. Stability correlates with frameshifting over its entire range, which suggests that stability is an important determinant of the probability of translation of the rephased complex.

Effects of the nucleotide 3' to an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1.

Rates of ribosomal selection of both release factor 1 (RF1) and a suppressor tRNA (Su7C33) were studied at an amber codon at which the 3' neighbor was permuted. Rates of RF1 selection vary 2.6-fold among contexts. The 3' neighbor-dependent variation of RF1 action correlates very strongly with the non-random frequencies of 3' neighbors at UAG terminators (r = 0.97), which argues that the rate of RF1 selection is an important determinant 3' neighbor choice at termination codons. The data are consistent with a model for RF1 selection in which RF1 makes a specific contact(s) to the 3' neighbor and that this interaction is most favorable to uridylic acid. Measured rates of Su7C33 selection vary fivefold among 3' contexts. We also develop a method to calculate rates of selection for other suppressors, based on the assumption that rates of RF1 selection at each 3' context can be generalized to other sites that have the same 3' neighbor. Rates for various suppressors appear to vary from two- to fivefold depending on the 3' neighbor. Generally, the rate of selection of suppressors at different contexts correlates with the stacking strength of the 3' neighbor as measured in vitro. The two- to fivefold range of 3' neighbor effects on rate of aminoacyl-tRNA selection is greater than that previously observed within sets of codons read by the same tRNA. It is suggested that the choice of codons to achieve favorable contexts may be more important than the choice of a common codon at some message sites.

Portable airway-suction systems: a comparison of performance.

UNLABELLED: A number of portable suction systems (powered either manually, pneumatically, or electrically) are available. We compared the performance of three electric systems (Laerdal Medical LSU, Laerdal Medical CSU, and Matrx Medical) and two manual systems (Vitalograph Emergency Aspirator and California Medical V-VAC) to wall suction set at maximum pressure of 300 torr [39.9 kPa]. METHODS: We determined the maximum pressure each system was capable of generating, and we measured the volume of imitation maple syrup each system at maximum pressure could suction within 5 seconds and the time required by each system at maximum pressure to suction 150 mL of syrup. In addition, we evaluated the life of each electric system's internal battery. RESULTS: All the electric systems were capable of generating suction pressure greater than 300 torr [39.9 kPa]. The amount of time required by the electric systems to suction 150 mL of syrup was not significantly different from that required by wall suction. In 5 seconds, wall suction suctioned a significantly greater volume of syrup than did the Matrx Medical system (p less than 0.05, ANOVA), but a significantly smaller volume of syrup than did the Laerdal Medical CSU system (p less than 0.05, ANOVA). The manual Vitalograph Emergency Aspirator was capable of generating 300 torr [39.9 kPa] pressure, but the California Medical V-VAC was not. Wall suction significantly outperformed both of the manual systems when volume of syrup suctioned in 5 seconds and time required to suction 150 mL of syrup were compared (P less than 0.05 ANOVA). All electric systems were capable of maintaining maximum suction greater than 15 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Rates of aminoacyl-tRNA selection at 29 sense codons in vivo.

We have placed aminoacyl-tRNA selection at individual codons in competition with a frameshift that is assumed to have a uniform rate. By assaying a reporter in the shifted frame, relative rates for association of the 29 YNN codons and their cognate aminoacyl-tRNAs were obtained during logarithmic growth in Escherichia coli. For five codons, three beginning with C and two with U, these relative rates agree with relative in vitro rates for elongation factor Tu-mediated aminoacyl-tRNA binding to ribosomes and subsequent GTP hydrolysis. Therefore, the frameshift assay probably measures this process in vivo. Observed rates for aminoacyl-tRNA selection span a 25-fold range. Therefore, the time required to transit different codons in vivo probably differs substantially. Codons very frequently used in highly expressed genes generally select aminoacyl-tRNAs more quickly than do rarely used codons. This suggests that speed of aminoacyl-tRNA selection is a significant factor determining biased use of synonymous codons. However, the preferential use of codons appears to be marked only for codons with the highest rates of aminoacyl-tRNA selection. Rapid selection in vivo is usually effected by elevation of the tRNA concentration for codons with moderate intrinsic speed (rate constant), not by choosing intrinsically fast codons. Despite a preference for high rate, there are quickly translated codons that are not commonly used, and common codons that are translated relatively slowly. Other factors are therefore more important than speed for some codons. Strong preference for rapid aminoacyl-tRNA selection is not observed in weakly expressed genes. Instead, there is a slight preference for slower aminoacyl-tRNA selection. The rate of aminoacyl-tRNA selection by a YNC codon is always greater than the rate of the corresponding YNU codon even though in many YNC/U pairs both codons react with the same elongation factor Tu/GTP/aminoacyl-tRNA complex. Thus, for these tRNAs, the differences between in vivo rate constants of tRNAs are dependent on the nature of anticodon base-pairing. However, no more general relationship is evident between codon/anticodon composition and rate of aminoacyl-tRNA selection. The frameshift method can be extended to all codons.

Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2.

It has been suggested that Escherichia coli release factor 2 (RF-2) translation is autoregulated. Mature RF-2 protein can terminate its own nascent synthesis at an intragenic, in-phase UGA codon, or alternatively, a +1 frameshift can occur that leads to completion of the RF-2 polypeptide. Translational termination presumably increases with RF-2 concentration, providing negative regulatory feedback. We now show, in lacZ/RF-2 fusions, that translation of a UAG codon at the position of the UGA competes with frameshifting, which proves one postulate of the translational autoregulatory model. We also identify a nearby sequence that is required for high-frequency frameshifting and suggest a constraint for the codon preceding the shift point. Both these sequences are incorporated into a model for frameshifting. Our measurements allow us to compute the relative rates in vivo of these reactions: release factor action, frameshifting and tRNA selection at an amber codon.

Reading frame selection and transfer RNA anticodon loop stacking.

Messenger RNA's are translated in successive three-nucleotide steps (a reading frame), therefore decoding must proceed in only one of three possible frames. A molecular model for correct propagation of the frame is presented based on (i) the measured translational properties of transfer RNA's (tRNA's) that contain an extra nucleotide in the anticodon loop and (ii) a straightforward concept about anticodon loop structure. The model explains the high accuracy of reading frame maintenance by normal tRNA's, as well as activities of all characterized frameshift suppressor tRNA's that have altered anticodon loops.

Base substitutions in the tRNA anticodon arm do not degrade the accuracy of reading frame maintenance.

We have examined the activities of a set of 34 site-directed mutants of tRNA Su7 for their ability to shift reading frame during translation of amber codons in vivo. The set includes variants at every position in the distal three base pairs of the anticodon stem and saturates the anticodon loop, with the exception of the anticodon itself. Most anticodon-stem mutations were made pairwise to preserve the secondary structure of that region. Variants of the Hirsh (A24) coding alteration were also tested. The mutations have varied and often dramatic effects on the ability of Su7 to act in translation, which indicates that they cause distortions of the codon-anticodon complex. However, none of the tested mutations affects the intrinsic accuracy of translocation, which we show to be very high. These results suggest that translocation must be independent of the conformational detail of the codon-anticodon complex and stand in contrast to frameshifts that occur when tRNAs misread codons. We suggest that when the tRNA is properly paired to the codon, translocation proceeds normally. Thus, we conclude that selection of a cognate tRNA ensures highly accurate reading frame maintenance. As a corollary, inefficient amber suppressors are not inefficient because they frameshift. Instead, they are likely to fail because a release factor translates the amber codon.

Transcription of Bacillis subtilis plasmid pBD64 and expression of bacteriophage SPO1 genes cloned therein.

Plasmid pBD64, a vector which is useful for cloning in Bacillis subtilis (T. J. Gryczan, A. G. Shivakumar, and D. Dubnau (1980), J. Bacteriol. 141, 246-253), has at least three substantial transcription units. Two of these include the single EcoRI, XbaI, and BamHI sites, while the other includes the single BglII site. Each of these transcripts was synthesized in the counterclockwise direction, relative to the pBD64 restriction map. No transcripts were detected in the opposite direction. Infection by bacteriophage SPO1 caused a substantial decrease in each of these transcripts. No new pBD64 transcripts were detected during SPO1 infection. Various SPO1 genes, cloned at several of these pBD64 sites, were tested for expression by observing their capacity to complement SPO1 mutants. Several middle and late genes were expressed substantially, regardless of the orientation in which the fragments were inserted. Since transcription from the vector could cause expression only in one orientation, this argues that the necessary transcription originated at SPO1 promoters, and, thus, that SPO1 middle and late promoters can be active in thymine-containing DNA.

Cloning and mapping of the SPO1 genome.

Many of the XbaI, EcoRI, KpnI, and BglII fragments of bacteriophage SPO1, accounting for about 65% of the genomic sequences, were cloned in Bacillus subtilis. Four of the EcoRI fragments were specifically refractory to cloning in both Escherichia coli and B. subtilis, probably because of expression of deleterious genes carried on the SPO1 fragments. To permit complete identification of the regions cloned, the SPO1 restriction map has been extended to include the XbaI fragments and the previously unmapped KpnI fragments. Markers for 26 of the 39 known genes have been located on specific cloned fragments, permitting more precise determination of the positions of most of the genes. One cloned SPO1 fragment was inhibitory to SPO1 development.