Total chemical synthesis and expression in Escherichia coli of a maize glutathione-transferase (GST) gene.

We have constructed a totally synthetic gene encoding a maize glutathione S-transferase (GST I). This gene, composed of 1320 nucleotides (nt) (660 bp), was assembled from only 16 synthetic oligodeoxynucleotides (average length 83 nt), using an efficient one-step annealing/ligation protocol. Sequencing was performed to verify the authenticity of the final assembled gene. Significantly, not a single mutation was found in either of the two constructs sequenced, indicating a remarkably low mutation frequency. The synthetic gene was introduced into Escherichia coli where it was successfully expressed. The biological activity of the GST I enzyme produced in E. coli was monitored by assaying bacterial extracts for the ability to conjugate [14C]atrazine in the presence of glutathione. This biologically active synthetic GST1 gene can now be introduced into plants to assess its ability to confer tolerance to the triazine class of herbicides.

Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene.

We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.

Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone alpha-subunit.

The glycoprotein hormones are a family of alpha beta heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone beta-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the alpha-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human alpha-subunit leader sequence and the secreted portion of the bovine alpha-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine alpha-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) beta-subunit resulted in the secretion of a functional alpha beta heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine alpha-, human alpha-, or hCG beta-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG beta-subunit and that formation of this epitope requires a change in conformation of the beta-subunit when it combines with the alpha-subunit.

Comparing 16 PF scores for two groups of offenders.

The 16 PF scores for 678 male offenders in a diagnostic and receiving center were compared with scores for 891 male offenders in penal institutions by t tests for independent means. Significant differences were obtained for 13 of the 16 primaries included in the 16 PF. The Penitentiary group scored significantly higher than the Reception Center group on the primaries, A, I, L, M, O, Q1, and Q4. Conversely, the Reception Center group scored significantly higher than the Penitentiary group on the primaries, B, C, F, G, N, and Q3.

Verbal/performance splits in inmates assessed with the Multidimensional Aptitude Battery .

A series of tables were created to examine the phenomenon of the overrepresentation of verbal/performance splits among incarcerated felons. Multidimensional Aptitude Battery (MAB; Jackson, 1984) scores were obtained for 1,225 adult male felons referred to a reception and diagnostic center for evaluation. The findings suggest that there is a significant, consistent overrepresentation of verbal/performance splits, with the performance score usually higher, among incarcerated male felons. The data further suggest that this phenomenon is not linked directly to age, reading level, or general level of intelligence.

Personality and intellectual characteristics of adult male felons as a function of offense category.

A step-wise discriminant function analysis identified demographic, personality, and intellectual variables that differentiate categories of felony offenders. Male inmates (N = 766) were categorized according to their most recent offense (Person, Property, Drug, or Indecent Liberties). Each completed the 16PF and the Multidimensional Aptitude Battery. A significant discriminant function was obtained, with loadings on the variables of age, substance abuse history, and the 16PF factors of Boldness and Intelligence. The function correctly classified 42.6% of the inmates into their offense category.

Factor structure of the Millon Clinical Multiaxial Inventory (MCMI) with an offender sample.

One thousand two hundred inmates were given the Millon Clinical Multiaxial Inventory (MCMI) in a midwestern reception and diagnostic center. Two groups of 600 were randomly divided, and their test results were subjected to a principal components factor analysis. Four factors were derived in both groups and were similar, indicating successful cross-validation. Three of the four factors bore similarity to factors found in other samples (drug abusers, psychiatric population, Viet Nam veterans), and the fourth was unique to the offender population.

Further evidence concerning motivational distortion on the Sixteen Personality Factor primaries by male felons.

A multiple regression analysis was used to determine the susceptibility of the 16 Personality Factor Test (16PF) to faking for a sample of male felons. The study is a replication of an earlier study of a similar sample. Motivational distortion (MD) correlated significantly with the 16PF primary scores. The relationship was most evident when the structure coefficients rather than the beta weights were analyzed. The findings were consistent with the previous results which indicated a fairly high degree of support for the MD corrections provided in the manual. An important exception was that Dominance (E) was suppressed by individuals from both samples when MD was present.

A combined factor analysis of the Millon Clinical Multiaxial Inventory and the MMPI in an offender population.

The Millon Clinical Multiaxial Inventory (MCMI) is a recently introduced instrument designed to measure both symptomatology and long-standing patterns of personality disorder in clinical populations. Because it is designed to assess clients who are similar to those with whom the MMPI is often used (e.g., adjudicated offenders), a question may arise as to whether the two instruments measure the same aspects of clients' function. This study investigated this question by conducting a combined factor analysis of the two instruments in a criminal offender population (N = 2,245). Results indicated that although there are important areas of overlap between the two instruments, each also contains unique sources of variance. The results are interpreted as supporting the use of both instruments as part of an objective assessment battery, as has been suggested by several authors.

Organochlorine pesticide residues in human milk samples from women living in Northwest and Northeast Mississippi, 1973-75.

Organochlorine pesticide analyses were performed on human milk samples obtained from 34 women living in the Mississippi Delta, a high pesticide usage area, and from six women living in Starkville, Mississippi, a low pesticide usage area. Nine women collected samples before and after their babies had nursed so that fat levels and sigma DDT levels could be compared on whole milk and milk fat bases. sigma DDT values were independent of collection time if calculated on a milk fat basis, but not if calculated on a whole milk basis. Thus, the most consistent indicator of DDT residues were values calculated on a milk fat basis. Residue levels for p,p'-DDE, p,p'-DDT, and sigma DDT were significantly different (P less than 0.01) in samples from the two areas. Residues of o,p'-DDT, beta-BHC, and oxychlordane in milk samples from women living in the high pesticide usage area also were significantly different (P less than 0.05). A mean value of 19.17 ppm sigma DDT, found in the milk fat of samples from the high pesticide usage area, is the highest ever reported. Samples from the low pesticide usage area contained a mean level of 2.36 ppm sigma DDT.

Rotational symmetry in ribonucleotide strand requirements for binding of HIV-1 Tat protein to TAR RNA.

Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge.

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.

Design and synthesis of RNA miniduplexes via a synthetic linker approach. 2. Generation of covalently closed, double-stranded cyclic HIV-1 TAR RNA analogs with high Tat-binding affinity.

We recently developed an approach which allows rapid generation of short, double-stranded oligonucleotides whereby one end of the duplex was joined and stabilized by a synthetic linker of specific design (miniduplexes)(6). Model miniduplexes based on the HIV-1 TAR RNA hairpin were shown to be thermodynamically stable and good substrates for binding by the HIV-1 Tat protein which normally bind to natural TAR (6). In this study, we have extended our studies to the design, synthesis and analysis of the binding properties of covalently closed, double-stranded, cyclic RNA miniduplexes. A strategy using automated chemical synthesis and T4 RNA ligase-catalyzed cyclization was employed to generate cyclic oligoribonucleotides. When both ends of a shortened, wild-type TAR RNA stem (9 bp) were covalently linked through either nucleotidic loops (4-6 nt) or synthetic linkers (derivatized from hexaethylene glycol), the resulting cyclic TAR RNA analogs were good substrates for binding by both Tat-derived peptide or full-length Tat protein. Interestingly, the cyclic TAR analogs failed to show any binding if the synthetic linker was reduced in length (e.g. derivatized from triethylene glycol), although such linkers are acceptable in the hairpin-shaped miniduplexes series (6). This implies that RNA conformational changes are required for Tat binding and that these changes are restricted in certain cyclic variants. Our findings suggest that covalently-closed nucleic acid miniduplexes may be useful both to study nucleic acid-protein interactions as well as to provide a basis for therapeutic intervention as transcription decoys.

Design and synthesis of RNA miniduplexes via a synthetic linker approach.

Double-stranded oligodeoxyribonucleotides or single-stranded oligoribonucleotides with specific secondary structure have been proposed as potential antagonists to target nucleic acid-binding proteins (the sense approach). A major limitation of this strategy is that these derivatives are generally considered to be too large for pharmaceutical applications. We have developed a synthetic linker approach whereby nucleic acid duplexes of a much smaller size (miniduplexes) can be generated directly from a standard oligonucleotide synthesis. In this approach, four synthetic linkers (derivatized respectively from 1,9-nonanediol, triethylene glycol, 1,3-propanediol, and hexaethylene glycol) of different length and hydrophobicity were designed and incorporated into a model RNA molecule based on the TAR stem-loop structure of HIV-1. Their thermal stabilities were evaluated by measuring denaturation profiles (Tm measurements). These linker-derivatized RNA molecules were then assessed for their ability to bind to either a full-length protein (HIV-1 Tat protein) or a short peptide (Tat-derived peptide) through RNA mobility shift assays. Results from this study indicate that such modified miniduplex structures retain full binding activity relative to that of the wild-type sequence (Kd values), while Tm values were increased by 24-31 degrees C compared to an open duplex of the same length. This system provides a new direction in the use of nucleic acid miniduplexes as a novel class of oligonucleotide analogues for both fundamental research and possible therapeutic applications.

Random mutagenesis of the human immunodeficiency virus type-1 trans-activator of transcription (HIV-1 Tat).

A new method is described for the direct construction of randomly mutagenized genes by applying the polymerase chain reaction (PCR) to an oligonucleotide synthesized using doped nucleotide reservoirs. We have demonstrated the utility of this method by generating a library of mutant HIV-1 tat genes. Several arbitrarily selected, inactive tat clones were sequenced to evaluate the extent of the mutagenesis. Moreover, fourteen recombinants encoding varying levels of transcriptional trans-activator activity were isolated by transient transfection of sub-library pools into a HeLa cell line bearing an HIV-LTR-chloramphenicol acetyltransferase (CAT) reporter gene. Sequence data revealed a spectrum of alterations including nucleotide substitutions, insertions, and deletions, suggesting that mutations arose from both the doped DNA synthesis and the subsequent PCR 'rescue' of full-length product. Sequence comparison between inactive and active Tat clones revealed a selection pressure against amino-acid substitutions within the N-terminal domains of Tat, indicating the importance of this region to trans-activation competence. In addition, single and double missense mutations within the basic-rich, TAR RNA-binding domain were seen to be tolerated within active Tat clones.

Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone beta subunit chimeras and mutants.

The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common alpha subunit but differ in their hormone-specific beta subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) beta subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LH beta-CG beta chimeras appeared to fold similar to hCG beta, since they combined with hCG alpha and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg8-Arg10,Gly47-Ala51, and Gln89-Leu92 form a major epitope region and appear to be adjacent to each other on the surface of hCG beta. Gly47-Ala51 and Gln89-Leu92 are recognized by dimer-specific mAbs while Arg8-Arg10 is recognized by mAbs which have highest affinity for the free beta subunit. These observations suggest that the conformation of this region of the beta subunit changes when the alpha and beta subunits combine. Residues which are C-terminal of Asp112 form a second epitope domain. mAbs to the third domain distinguish hCG beta and hLH beta by the presence of Asn77 in hCG beta and can be detected after hCG binds to receptors. These findings were used to develop a model of hCG beta which predicts the locations of these residues and their positions relative to the alpha subunit and receptor interfaces.