Biotechnology and the chicken B cell line DT40.

Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin (Ig) gene by gene conversion and somatic hypermutation. This machinery can be directed to almost any gene inserted into the Ig locus. Enormously diverse protein libraries of any gene of interest can be quickly generated in DT40 by utilizing random shuffling of complex genetic domains (gene conversion) and by the introduction of novel non-templated genetic information (random mutagenesis). The unique characteristics of the chicken cell line DT40 make it a powerful in-cell diversification system to improve proteins of interest within living cells. One essential advantage of the DT40 protein optimization approach is the fact that variants are generated within an in-cell system thus allowing the direct screening for desired features in the context of intracellular networks. Utilizing specially designed selection strategies, such as the powerful fluorescent protein technology, enables the reliable identification of protein variants exhibiting the most desirable traits. Thus, DT40 is well positioned as a biotechnological tool to generate optimized proteins by applying a powerful combination of gene specific hypermutation, gene conversion and mutant selection.

Increased transcription levels induce higher mutation rates in a hypermutating cell line.

Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81.

An experimental solution for the Luria-Delbrück fluctuation problem in measuring hypermutation rates.

A cell line harboring all trans-acting elements necessary for hypermutation was transfected with a plasmid harboring the major cis-acting elements plus a green fluorescent protein gene containing a premature chain-termination codon. Transfected cells do not fluoresce unless the stop codon reverts. When a sizable cell population is purged of revertants by sorting, the frequency of mutants increases linearly with time, and there is no Luria-Delbrück fluctuation effect. Moreover, as mutant frequencies seemed to vary less than cell numbers in replicate cultures, it is suggested that hypermutation might not be coupled closely to cell division.

Hypermutation targets a green fluorescent protein-encoding transgene in the presence of immunoglobulin enhancers.

Hypermutation introduces point mutations into the gene segments encoding immunoglobulin variable regions at a rate that is a million-fold higher than the spontaneous mutation rate in most of the genome. Because Ig enhancers are required to target hypermutation, transcription appears to play a critical role for the hypermutation mechanism. We have developed a novel system for detecting mutations that enables us to determine the influence of expression levels on the mutability of a transgene. This system utilizes a green fluorescent protein receptor gene and the powerful enumeration and quantification properties of flow cytometry. We have tested this system with several constructs bearing Ig enhancers in cell lines with active and inactive hypermutation systems.

The Ig mutator is dependent on the presence, position, and orientation of the large intron enhancer.

Hypermutation at the Ig loci is confined to the area between the promoter and the intronic enhancer, which includes the rearranged variable region gene segment. We identified factors that contribute to the site-specificity at the heavy chain locus. We found that distance from both the promoter and the intronic enhancer is crucial in hypermutation. The presence of the enhancer is required, and, in contrast to its definition for transcriptional activity, its effect is orientation-sensitive.

Critical test of hot spot motifs for immunoglobulin hypermutation.

In hypermutation at the immunoglobulin loci, some bases are much more mutable than others. The increased mutability of the hot spots has been attributed to their being embedded in short sequence motifs. Among the suggested motifs are palindromes, TAA and RGYW (i.e. A/G G C/T A/T). We have tested these proposed motifs in a transfection system in vitro, which ordinarily uses the hypermutable stop codon TAG. The stop codon TAA is not hypermutable in our system, even when embedded in the pentamer and hexamer palindromes TAATA and ATTAAT; in fact, the revertants isolated were due to deletions. Single or double base changes in an RGYW motif containing a hypermutable stop codon result in a reduction of one order of magnitude or more in point mutation frequency. When the nonamer GACTAGTAT, which includes the same RGYW motif, was moved over hundred base pairs upstream, hypermutability was reduced by an order of magnitude. Thus, while RGYW apparently is a hypermutability motif, it cannot be the sole determinant of mutability.

Translatable immunoglobulin germ-line transcript.

During B cell differentiation, the functional genes encoding immunoglobulin (Ig) heavy (H) and light (L) chains are generated by two rearrangement processes--VDJ rearrangement generates the exon encoding the Ig variable (V) regions, and the class switch reconstructs a rearranged IgH gene by exchanging the segment encoding the constant (C) region, which determines the Ig class. Both types of rearrangement are preceded by transcripts originating from a transcriptional start site 5' of the I exon, which is then spliced to the C exons. These germ-line transcripts, which are thought to be necessary for the initiation of both types of rearrangement, are said to be sterile. We demonstrate here that the mu germ-line transcript is translatable into a polypeptide chain, to which we assign the symbol psi. Thus, protein products of these transcripts might be part of or signal to the recombinases that catalyze Ig gene rearrangement.

An immunoglobulin mutator that targets G.C base pairs.

Hypermutation can be defined as an enhancement of the spontaneous mutation rate which the organism uses in certain types of differentiated cells where a high mutation rate is advantageous. At the immunoglobulin loci this process increases the mutation rate > 10(5)-fold over the normal, spontaneous rate. Its proximate cause is called the immunoglobulin mutator system. The most important function of this system is to improve antibody affinity in an ongoing response; it is turned on and off during the differentiation of B lymphocytes. We have established an in vitro system to study hypermutation by transfecting a rearranged mu gene into a cell line in which an immunoglobulin mutator has been demonstrated. A construct containing the mu gene and the 3' kappa enhancer has all the cis-acting elements necessary for hypermutation of the endogenous gene segments encoding the variable region. The activity of the mutator does not seem to depend strongly on the position of the transfected gene in the genome. The mutator is not active in transformed cells of a later differentiation stage. It is also not active on a transfected lacZ gene. These results are consistent with the specificity of the mutator system being maintained and make it possible to delineate cis and trans mutator elements in vitro. Surprisingly, the mutator preferentially targets G-C base pairs. Two hypotheses are discussed: (i) the immunoglobulin mutator system in mammals consists of several mutators, of which the mutator described here is only one; or (ii) the primary specificity of the system is biased toward mutation of G-C base pairs, but this specificity is obscured by antigenic selection.

Enhancers of hypermutation.

Hypermutation at the immunoglobulin (Ig) loci increases the mutation rate more than 10(5)-fold over the normal, spontaneous rate. We studied two kinds of cis-acting elements - 3' enhancers and promoters - in a system in which a gene encoding the mu heavy (H) chain (Igm) is transfected in vitro into a cell line with an active Ig mutator. A construct containing a rearranged Igm gene requires the 3' H enhancer for hypermutation at a rate comparable with the one at the endogenous gene segment encoding the H chain variable region (V). Without the 3' enhancer, the basal mutational activity is much lower, but still higher than the normal, spontaneous mutation rate. Replacement of the 3' H enhancer by atopic elements of similar function also supports full hypermutation. Even though these 3' elements are defined as transcriptional enhancers, they do not seem to increase hypermutation via an increase in the rate of transcription. Replacement of the endogenous promoter by the tk promoter slightly increases hypermutability of the construct; thus, no specific sequences in the Ig promoter are likely to target hypermutation.

Effect of deletions at structural domains of group II intron bI1 on self-splicing in vitro.

Some group II introns can undergo a protein-independent splicing reaction with the basic reaction pathway similar to nuclear pre-mRNA splicing and the catalytic functions of some of the structural components have been determined. To identify further functional domains, we have generated an ensemble of partial and complete deletions of domains I, II, III and IV of the self-splicing group II intron bI1 from yeast mitochondria and studied their effects on the splicing reaction in vitro. Our results indicate that domains II and IV, which vary considerably in length and structure among group II introns, do not play a direct role in catalysis but mainly help to ensure the proper interaction between upstream and downstream catalytically active structural elements. Deletions of sub-domains of domain I and domain III indicate that these elements are involved in 5' cleavage by hydrolysis and in a reaction in trans (exon reopening), and that this function can be inhibited without affecting the normal 5' cleavage by transesterification. Yet, we infer that the helical structures affected by the mutational alterations might not contribute to this reaction mode per se but that changes within local secondary structures perturb the internal conformation of the ribozyme. Furthermore, we have designed an abbreviated version of intron bI1, with a length of 542 nucleotides, which is still catalytically active.