Harry W. Jarrett

Cloning and disruption of the yeast C-8 sterol isomerase gene

The yeastERG2 gene codes for the C-8 sterol isomerase, an enzyme required for the isomerization of the δ8 double bond to the δ7 position in ergosterol biosynthesis. TheERG2 gene was cloned by complementation of a C-8 sterol isomerase mutant strain (erg2). The complementing region of DNA required to restore ergosterol synthesis toerg2 was limited to a 1.0 kbStuI-BglII fragment. In order to determine whether theERG2 gene was essential for yeast viability, aLEU2 gene was inserted into theNdeI site (made blunt) of this 1.0 kb fragment. Transformation of a wild type diploid strain with theERG2 substituted DNA resulted in the generation of viable haploids containing theerg2 null allele (erg2–4∶∶Leu2). These results suggest that the C-8 sterol isomerase activity is not essential for yeast cell viability. This disruption represents the second ergosterol biosynthetic gene in the distal portion of the pathway to be disrupted without adversely affecting cell viability.

Affinity chromatography with nucleic acid polymers

Column chromatography utilizing polynucleotides immobilized on solid support is reviewed. This form of affinity chromatography is used for the isolation of polynucleotides and polynucleotide binding proteins, and to a lesser extent for analysis. Several specific applications within these categories have been widely used in the biomedical sciences. Poly(A) mRNA is routinely isolated using oligo(dT) or oligo(dU) supports. Many DNA binding proteins, including transcription factors, restriction endonucleases, and proteins involved in DNA repair, replication, recombination, and transposition have been purified using DNA affinity chromatography. Recently, DNA supports suitable for use in high-performance liquid chromatography have been described and utilized. The current usage of DNA affinity chromatography is reviewed and potential future uses for this technology are speculated upon.

Oligonucleotide trapping method for purification of transcription factors.

A new oligonucleotide trapping method in which a decameric oligonucleotide (AC)5 coupled to Sepharose is used to trap a complex of a transcription factor and its corresponding specific DNA element is described. The concentration of DNA element used in the trapping method was very low (50 nM) and hence discouraged binding of nonspecific proteins. We have shown that this method gives higher purity for green fluorescent protein CAAT enhancer binding chimeric protein (GFP-C/EBP) than the biotin-avidin method. We have also shown that the oligonucleotide trapping method has a capacity close to 95% of the theoretical capacity, which is significantly greater than the 15% capacity obtained with conventional DNA affinity columns. The purity of GFP-C/EBP obtained using a low concentration of the oligonucleotide in our trapping method is three-fold higher (3,668- versus 1,028-fold) than that obtained by conventional DNA affinity chromatography and the yield was also higher (36% versus 24%). Highly purified transcription factor B3 is obtained from Xenopus egg crude extract using the oligonucleotide trapping method as the only purification.

Affinity purification of DNA-binding proteins

The focus of this review is on DNA affinity chromatography, which is the most powerful tool for purification of DNA binding proteins. The use of nonspecific-, sequence specific- and single stranded-DNA affinity columns in purification of various DNA binding proteins is discussed. The purification strategies for transcription factors, restriction enzymes, telomerases, DNA and RNA polymerase and DNA binding antibodies are described. Different applications of DNA affinity chromatography are presented.

Novel DNA-sepharose purification of the FadR transcription factor

A DNA sequence bound by the FadR transcription factor of Escherichia coli was covalently attached to Sepharose by two different approaches: by chemical coupling or by template-directed enzymatic synthesis using a DNA polymerase. The two kinds of DNA-Sepharose were packed into small columns and used for the purification of the FadR protein; chromatography was without using competitor DNA and the supports contained single-copy, non-repetitive DNA sequences. Comparison showed that the enzymatically prepared support, while having less bound DNA, bound more FadR protein than did the chemically prepared support. This probably results from the lack of detrimental DNA modification by the gentle enzymatic procedure. The chemically prepared support was of lower capacity but yielded purer FadR protein when compared under the same elution conditions. This may be explained by the simpler DNA sequence which could be coupled chemically; less contaminating proteins were bound by the simpler DNA sequence. However, the enzymatically prepared support could also yield comparable purity if the protocol was modified to include additional washes with salt containing buffers. In all cases, FadR was eluted from the DNA using high-salt (0.8 M) mobile phase; ligand-specific elution of FadR using a fatty acyl-coenzyme A thiol ester was ineffective. Affinity chromatography on DNA-Sepharose provided a more rapid, simple purification of FadR than conventional purification techniques and yielded biologically active protein.

Cyanogen bromide activation and coupling of ligands to diol-containing silica for high-performance affinity chromatography optimization of conditions.

To obtain silica supports for high-performance affinity chromatography, a method of preparing CNBr-activated diol-silica under anhydrous conditions was developed. Activation of the silane-derived hydroxyls with cyanogen bromide and triethylamine was optimized and demonstrated to efficiently couple several amino ligands (tryptophan, 6-aminohexyl-Cibacron Blue, and DNA). Sonication and vacuum degassing, a procedure used to remove air from the silica beads, increased activation. Coupling of an amino ligand under slightly basic conditions (pH 8.0) gave the highest yield. The linkage between the immobilized ligands and silica was stable from pH 2-10. Anhydrous acetone was the most effective solvent for activation but dimethylformamide and 2-propanol were also good choices. The high-performance affinity chromatography columns obtained by coupling sequence-specific DNA binding sequences for Lac repressor-beta-galactosidase fusion protein were compared to affinity columns obtained by coupling the same DNA element to Sepharose beads; 5 microm silica gave the best performance.

Comparative studies on chemically and enzymatically coupled DNA–Sepharose columns for purification of a lac repressor chimeric fusion protein

The length of a DNA sequence attached to an affinity chromatography column affects column retention of transcription factors. Even when unrelated sequences such as a poly(A):poly(T) tail are included in a DNA sequence, transcription factors such as the lac repressor are bound more tightly by the column. The position of the additional sequences is also important. To compare coupling procedures, an identical DNA sequence was covalently attached to Sepharose by chemical coupling or produced enzymatically by template driven enzymatic primer extension. These two types of supports, containing the O1 operator sequence bound by lac repressor, were packed into identical columns and compared by purification of a lac repressor-beta-galactosidase fusion protein. We found that the purity and yield of proteins eluted from the two columns were similar. Overall, the results suggest that there is no significant advantage to either type of support for the purification of some proteins. The study revealed a potentially important effect of the length of DNA sequences on column selectivity.

Activation of enzymes by calmodulins containing intramolecular cross-links.

We have reacted calmodulins containing cysteines substituted at positions 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate intramolecularly cross-linked proteins termed BMHCM or BMHCM1, respectively. Reactions were also performed with N-ethylmaleimide (NEM) in place of BMH to generate corresponding S-ethylsuccinimidylated proteins termed NEMCM or NEMCM1. The abilities of these proteins to activate plant NAD kinase, erythrocyte Ca(2+)-ATPase and bovine brain calcineurin activities were assessed. The BMH- or NEM-reacted proteins activate calcineurin activity as does control calmodulin. Kact values for Ca(2+)-ATPase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in Vmax values. Activation of this enzyme by NEMCM or NEMCM1 is not different from the control. In NAD kinase activation experiments BMHCM and BMHCM1 are associated with a 10 to 20-fold increase in Kact values and a 60-75% reduction in Vmax values relative to the control. NEMCM1 is not associated with any apparent changes in NAD kinase activation, however, NEMCM is associated with a 10-fold increase in the Kact value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased Kact value, implying that this change is due to interactions between S-(ethylsuccinimido)cysteines at positions 3 and 146. In conclusion, cross-linking and associated distortions in the structure of calmodulin appear to have little or no effect on activation of calcineurin enzyme activity. However, bending in the central helix and/or steric restrictions associated with cross-linking increase significantly the Kact value for Ca(2+)-ATPase and NAD kinase activation, and dramatically reduce maximal activation of NAD kinase activity.

DNA-support coupling for transcription factor purification: Comparison of aldehyde, cyanogen bromide and N-hydroxysuccinimide chemistries

Purification of transcription factor IIIA on internal control region DNA coupled to aldehyde-silica is described and compared with purification on cyanogen bromide-activated Sepharose and Bio-Rad Affi-Gel-10. The Affi-Gel support results in mixed-mode chromatography; both ion-exchange and affinity modes contribute. Coupling DNA to aldehyde-silica is advantageous in that it has no ion-exchange properties and performs as well as DNA coupled to CNBr-activated Sepharose. Purification of lac repressor on aldehyde-silica, and CAAT enhancer binding protein on Affi-Gel also shows the advantages of a neutral support and the disadvantages of mixed-mode chromatography for transcription factor purification. Aldehyde-silica couples to alkylamines and to the amines of adenine, guanine, and cytosine nucleoside bases. Reaction occurs with either single- or double-stranded DNA, although it is less efficient with the latter. Overall, the results demonstrate that predominantly neutral coupling chemistries, such as aldehyde or CNBr-mediated coupling, have distinct advantages for transcription factor purification. Since the CNBr chemistry has not yet been applied to silica supports, aldehyde-silica coupling is currently the most attractive method for DNA affinity HPLC.

Preparation of cDNA-silica using reverse transcriptase and its DNA sequence determination.

A new method for producing macroporous silica (suitable for high-performance liquid chromatography) with covalently attached DNA is presented. The method uses (dT)18-silica as a primer, annealed to a poly(A)-RNA template, which is then transcribed using reverse transcriptase. The RNA template is eluted and single-stranded cDNA-silica is recovered. The cDNA-silica can be sequenced using the dideoxy method. These methods provide a facile method for producing cDNA-silica of demonstrable authenticity and provide a unique approach to DNA and RNA sequencing.