Weak Sequence Homology Research Articles

HU‐α binds to the putative double‐stranded DNA mimic HI1450 from Haemophilus influenzae

Recently, the solution structure of the hypothetical protein HI1450 from Haemophilus influenzae was solved as part of a structure-based effort to understand function. The distribution of its many negatively charged residues and weak structure and sequence homology to uracil DNA glycosylase inhibitor (Ugi) suggested that HI1450 may act as a double-stranded DNA (dsDNA) mimic. We present supporting evidence here and show that HI1450 interacts with the dsDNA-binding protein HU-alpha. The interaction between HI1450 and HU-alpha from H. influenzae is characterized using calorimetry and NMR spectroscopy. HU-alpha binds to HI1450 with a K(d) of 3.0 +/- 0.2 microM, which is similar in affinity to its interaction with dsDNA. Chemical shift perturbation data indicate that the beta1-strand of HI1450 and neighboring regions are most directly involved in interactions with HU-alpha. These results show that HI1450 and its structural homolog, Ugi, use similar parts of their structures to recognize DNA-binding proteins.

Mouse Sir2 Homolog SIRT6 Is a Nuclear ADP-ribosyltransferase

Members of the Sir2 family of NAD-dependent protein deacetylases regulate diverse cellular processes including aging, gene silencing, and cellular differentiation. Here, we report that the distant mammalian Sir2 homolog SIRT6 is a broadly expressed, predominantly nuclear protein. Northern analysis of embryonic samples and multiple adult tissues revealed mouse SIRT6 (mSIRT6) mRNA peaks at day E11, persisting into adulthood in all eight tissues examined. At the protein level, mSIRT6 was readily detectable in the same eight tissue types, with the highest levels in muscle, brain, and heart. Subcellular localization studies using both C- and N-terminal green fluorescent protein fusion proteins showed mSIRT6-green fluorescent protein to be a predominantly nuclear protein. Indirect immunofluorescence using antibodies to two different mSIRT6 epitopes confirmed that endogenous mSIRT6 is also largely nuclear. Consistent with previous findings, we did not observe any NAD+-dependent protein deacetylase activity in preparations of mSIRT6. However, purified recombinant mSIRT6 did catalyze the robust transfer of radiolabel from [32P]NAD to mSIRT6. Two highly conserved residues within the catalytic core of the protein were required for this reaction. This reaction is most likely mono-ADP-ribosylation because only the modified form of the protein was recognized by an antibody specific to mono-ADP-ribose. Surprisingly, we observed that the catalytic mechanism of this reaction is intra-molecular, with individual molecules of mSIRT6 directing their own modification. These results provide the first characterization of a Sir2 protein from phylogenetic class IV.

Crystal Structure of the tRNA 3′ Processing Endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.

Recognizing and Defining True Ras Binding Domains I: Biochemical Analysis

Common domain databases contain sequence motifs which belong to the ubiquitin fold family and are called Ras binding (RB) and Ras association (RalGDS/AF6 Ras associating) (RA) domains. The name implies that they bind to Ras (or Ras-like) GTP-binding proteins, and a few of them have been documented to qualify as true Ras effectors, defined as binding only to the activated GTP-bound form of Ras. Here we have expressed a large number of these domains and investigated their interaction with Ras, Rap and M-Ras. While their (albeit weak) sequence homology suggest that the domains adopt a common fold, not all of them bind to Ras proteins, irrespective of whether they are called RB or RA domains. We used fluorescence spectroscopy and isothermal titration calorimetry to show that the binding affinities vary over a large range, and are usually specific for either Ras or Rap. Moreover, the specificity is dictated by a set of key residues in the interface. Stopped-flow kinetic analysis showed that the association rate constants determine the different affinities of effector binding, while the dissociation rate constants are in a similar range. Manual sequence analysis allowed us to define positively charged sequence epitopes in certain secondary structure elements of the ubiquitin fold (beta1, beta2 and alpha1) which are located at similar positions and comprise the hot spots of the binding interface. These residues are important to qualify an RA/RB domain as a true candidate Ras or Rap effector.

The HMG-I/Y-related Protein p8 Binds to p300 and Pax2trans-Activation Domain-interacting Protein to Regulate thetrans-Activation Activity of the Pax2A and Pax2B Transcription Factors on the Glucagon Gene Promoter

p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8 in vitro, although the significance of such modification remains to be determined. Then a screening by the two-hybrid system, using p8 as bait, allowed us to identify the Pax2 trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B trans-activation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2-interacting protein PTIP to suppress its inhibition.

Isolation of a Novel Interleukin-1-inducible Nuclear Protein Bearing Ankyrin-repeat Motifs

We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IkappaB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor alpha nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-kappaB1/p50, NF-kappaB2/p52, C/EBPbeta, and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other IkappaB family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus.

Capsule essential for fatal tropical disease pathogen

Burkholderia pseudomallei causes melioidosis, an emerging disease in tropical areas such as South-east Asia and northern Australia, with a high fatality rate. It usually presents as a severe pneumonia or septicaemia, although subclinical infection is also seen, and relapses are common. Infection often responds poorly to antibiotic treatment, and alternative therapies are urgently required.The reservoir of B. pseudomallei is soil and stagnant water. The closely related species, B. thailandensis, is also found in these environments but is not pathogenic in humans. In a Syrian hamster model of septicaemia, the lethal dose that kills 50% of animals tested (LD50) of B. pseudomallei is ten organisms, whereas for B. thailandensis it is 106. Reckseidler et al. 1xDetection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant. Reckseidler, S.L et al. Infect. Immun. 2001; 69: 34–44Crossref | PubMed | Scopus (184)See all References1 have now exploited this difference, using subtractive hybridization to identify genes that are present in B. pseudomallei and not B. thailandensis, with the aim of identifying those necessary for virulence. One of the unique genes identified showed weak sequence homology to a Pseudomonas aeruginosa glycosyltransferase that is involved in capsule synthesis. Following this clue, the team then constructed a strain of B. pseudomallei with a mutation in this gene (wbpX). In the hamster model, the LD50 of the mutant was 3.5×105 organisms, thus demonstrating the role of wbpX in pathogenesis.As it was hypothesized that wbpX was also involved in capsule synthesis, the authors visualized the bacterial capsule using immunogold staining, and compared wild-type to mutant strains. The mutant strain was found to be deficient in type I O-polysaccharide (O-PS) moieties found on the cell surface. Further sequencing of the B. pseudomallei chromosome around wbpX identified a large cluster of genes, all of which are either predicted or proven to be necessary for type I O-PS synthesis or transport. These genes showed sequence homology to group 3 capsule genes found in other pathogens such as Neisseria meningitidis, Haemophilus influenzae, and Escherichia coli. Furthermore, this region has a G+C content that is unusually low for B. pseudomallei, suggesting it is encoded on a pathogenicity island that has been horizontally acquired from another bacteria. It is therefore possible that the acquisition of these genes has enabled B. pseudomallei to become a pathogen.Capsules often make good targets for bacterial vaccines. Before a vaccine to melioidosis can be developed, it will be necessary to prove that antibody to group 3 capsule is protective, and that protective antibodies are induced after vaccination. If successful, such a vaccine might be particularly useful for visitors to endemic countries, as well as preventing disease in high-risk populations and relapse in seropositive patients.

Characterization of the coral allene oxide synthase active site with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy: evidence for tyrosinate ligation to the ferric enzyme heme iron.

Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV--visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g = 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g = 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K(d) = 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.

Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame.

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.

Crystal structure of the surfactin synthetase-activating enzyme Sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily

The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the Sfp-CoA complex determined at 1.8 A resolution reveals a novel alpha/beta-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate.

Identification and expression analysis of two fungal cDNAs regulated by ectomycorrhiza and fruit body formation

Ectomycorrhiza formation is a complex developmental process that is still not well understood. To study this process, we identified genetic markers for mycorrhiza development by differential screening of a cDNA library obtained from fully developed Picea abies–Amanita muscaria mycorrhizas. Twenty-three cDNA clones were identified that showed significantly altered gene expression during the ectomycorrhizal interaction. A detailed analysis was performed for two fungal cDNA clones, SC13 and SC25, exhibiting the most pronounced differences. SC13 encodes a protein of 184 amino acid residues that shows no homology with any sequence in databases. It was highly expressed in non-mycorrhizal hyphae, whereas its expression was decreased at least 50-fold in mycorrhizas and fruit bodies. SC25 encodes a protein of 198 residues that shows weak sequence homology with extensin-like plant proteins. The expression of this gene was weak in non-mycorrhizal hyphae but approx. 30-fold higher in mycorrhizas and fruit bodies. Because the expression of both developmentally regulated fungal genes was identical for mycorrhizas and fruit bodies, a common regulation mechanism for both developmental processes is proposed.

Crystal structure of the bacterial cell-division protein FtsZ.

Bacterial cell division ends with septation, the constriction of the cell wall and cell membranes that leads to the formation of two daughter cells. During septation, FtsZ, a protein of relative molecular mass 40,000 which is ubiquitous in eubacteria and is also found in archaea and chloroplasts, localizes early at the division site to form a ring-shaped septum. This septum is required for the mechanochemical process of membrane constriction. FtsZ is a GTPase with weak sequence homology to tubulins. The nature of FtsZ polymers in vivo is unknown, but FtsZ can form tubules, sheets and minirings in vitro. Here we report the crystal structure at 2.8 A resolution of recombinant FtsZ from the hyperthermophilic methanogen Methanococcus jannaschii. FtsZ has two domains, one of which is a GTPase domain with a fold related to one found in the proteins p21ras and elongation factor EF-Tu. The carboxy-terminal domain, whose function is unknown, is a four-stranded beta-sheet tilted by 90 degrees against the beta-sheet of the GTPase domain. The two domains are arranged around a central helix. GDP binding is different from that typically found in GTPases and involves four phosphate-binding loops and a sugar-binding loop in the first domain, with guanine being recognized by residues in the central connecting helix. The three-dimensional structure of FtsZ is similar to the structure of alpha- and beta-tubulin.

Molecular Modeling of Immunoglobulin Superfamily Proteins: Predicting the Three Dimensional Structure of the Extracellular Domain of CTLA-4 (CD152)

The interactions between CD28/CTLA-4 (CD152) on T cells and their ligands CD80/CD86 on antigen presenting cells provide costimulatory signals critical for T cell activation. CD28/CTLA-4 and CD80/CD86 are members of the immunoglobulin superfamily (IgSF). CD28 and CTLA-4 both contain a single extracellular immunoglobulin (Ig) domain which binds CD80/CD86. Here we report modeling studies on the three-dimensional (3D) structure of the CTLA-4 binding domain. Since CTLA-4 displays only very weak sequence homology to proteins with known 3D structure, conventional modeling techniques were difficult to apply. Structure-oriented sequence comparison, consensus residue analysis, conformational searching, and inverse folding calculations were employed to aid in the generation of a comparative CTLA-4 model. Regions of high and low prediction confidence were identified, and the sequence-structure compatibility of the model was determined. Characteristics of the modeled structure, which resembles an Ig V domain, were analyzed, and the model was used to map N-linked glycosylation sites and residues critical for CTLA-4 function. The modeling approach described here can be applied to predict 3D structures of other IgSF proteins.

Limulus factor D, a 43-kDa protein isolated from horseshoe crab hemocytes, is a serine protease homologue with antimicrobial activity

A glycoprotein ( M r = 43 000) from horseshoe crab hemocytes with antimicrobial activity against Gram-negative bacteria was purified. The internal peptide sequences coincided exactly with the deduced amino acid sequence of a cDNA clone, designated limulus factor D, which was isolated by screening a hemocyte cDNA library with an anti-human plasminogen antibody. The open reading frame codes for a precursor of factor D of 394 amino acid residues, including an NH 2-terminal signal sequence. The COOH-terminal domain of factor D has significant sequence homology with the catalytic domain of mammalian serine proteases, in particular with human tissue plasminogen activator (32% identity), except for the substitution of Ser of the active site triad to Gly. Factor D has a unique NH 2-terminal domain with weak sequence homology with part of the mammalian interleukin-6 receptor α-chain. Factor D is likely to have an important role in host defense mechanisms.

NMR structure determination of tick anticoagulant peptide (TAP).

Tick anticoagulant peptide (TAP) is a potent and selective 60-amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo- and heteronuclear two-dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C-labeled, and 15N-labeled TAP provided nearly complete 1H sequence-specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY-derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa.

Identification and analysis of multigene families by comparison of exon fingerprints

Gene families are often recognised by sequence homology using similarity searching to find relationships, however, genomic sequence data provides gene architectural information not used by conventional search methods. In particular, intron positions and phases are expected to be relatively conserved features, because mis-splicing and reading frame shifts should be selected against. A fast search technique capable of detecting possible weak sequence homologies apparent at the intron/exon level of gene organization is presented for comparing spliceosomal genes and gene fragments. FINEX compares strings of exons delimited by intron/exon boundary positions and intron phases (exon fingerprint) using a global dynamic programming algorithm with a combined intron phase identity and exon size dissimilarity score. Exon fingerprints are typically two orders of magnitude smaller than their nucleic acid sequence counterparts giving rise to fast search times: a ranked search against a library of 6755 fingerprints for a typical three exon fingerprint completes in under 30 seconds on an ordinary workstation, while a worst case largest fingerprint of 52 exons completes in just over one minute. The short "sequence" length of exon fingerprints in comparisons is compensated for by the large exon alphabet compounded of intron phase types and a wide range of exon sizes, the latter contributing the most information to alignments. FINEX performs better in some searches than conventional methods, finding matches with similar exon organization, but low sequence homology. A search using a human serum albumin finds all members of the multigene family in the FINEX database at the top of the search ranking, despite very low amino acid percentage identities between family members. The method should complement conventional sequence searching and alignment techniques, offering a means of identifying otherwise hard to detect homologies where genomic data are available.

Similarity of three-dimensional microcrystals of detergent-solubilized (Na+,K+)-ATPase from pig kidney and Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum

Image analysis has been used to compare the projection structure of three-dimensional crystals of (Na+,K+)-ATPase from pig kidney and the Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. These crystals, formed from detergent-solubilized protein in the presence of 20% glycerol and at a low detergent to protein ratio, crystallize in nearly identical unit cells in the space group C2. Average cell dimensions for the Ca(2+)-ATPase were a = 166.8 +/- 4.5 A, b = 57.7 +/- 4.4 A, gamma = 90 degrees while those for the (Na+,K+)-ATPase were a = 166.2 +/- 3.8A, b = 54.25 +/- 3.5A, gamma = 90 degrees. Their projected structures at the resolution of 25-A resolution are indistinguishable. Thus, the (Na+,K+)-ATPase crystals appear to contain only the alpha chain of the alpha beta heterodimer found in native membranes. We conclude from this that the three-dimensional structure of the alpha chain of the (Na+,K+)-ATPase is very similar to that of the Ca(2+)-ATPase despite their relatively weak overall sequence homology.

Plant chitinases

Plant chitinases

Identification and characterization of a human herpesvirus 6 gene segment that trans activates the human immunodeficiency virus type 1 promoter.

Human herpesvirus 6 (HHV-6) is a lymphotropic herpesvirus, and in vitro, HHV-6 can productively infect many of the same cell types as can human immunodeficiency virus (HIV). Coinfection by both viruses in vitro can lead to both activation of the HIV promoter and acceleration of cytopathic effects. We have previously demonstrated that a large, 22.25-kb cloned HHV-6 fragment, pZVB70, can trans activate HIV promoter expression in vitro. In this study, we show that the pZVB70 fragment can trans activate the HIV promoter in human T-cell lines as well as in the monkey kidney cell line CV-1. The pZVB70 insert was digested with various restriction enzymes, and individual fragments were transfected into cells to test for their ability to trans activate the HIV promoter. By this method, we have identified a 1.8-kb subfragment, B701, that is involved in trans activation. Sequence analyses show that B701 potentially encodes a 143-amino-acid protein. This protein shares no homology with other herpesvirus proteins, such as ICP0 and ICP4, that have been shown to trans activate the HIV promoter. However, it shows weak sequence homology with the gene products encoded by the cytomegalovirus early US22 gene family, suggesting that the putative B701 protein may be an HHV-6 early regulatory protein. The 143-amino-acid coding sequence of B701 was cloned by polymerase chain reaction, and transfection of this construct into cells activated HIV promoter expression. The target site on the HIV promoter for the putative B701 protein is mapped to the NF-kappa B binding site. Our results suggest that the putative B701 protein may function by directly binding to the NF-kappa B site or may involve cellular factors, such as NF-kappa B, either directly or indirectly.

Antibody engineering using very long template-assembled oligonucleotides

We have previously constructed single-stranded DNA fragments up to 361 nucleotides long by the ligation of shorter synthetic fragments assembled on a closely homologous template and used them for site-directed mutagenesis. Here we have extended the utility of such long oligonucleotides for protein engineering in two ways. First oligonucleotides have been assembled on a template to which they show only relatively weak sequence homology (<70% identity), allowing conversion of genes encoding rather distantly related proteins. We have converted a gene encoding human light chain constant domain from the kappa to the lambda isotype, which share <40% amino acid identity. Second, libraries of random mutants were constructed within antibody variable domains with up to 20 targeted codons separated by up to 186 nucleotides. These libraries were created by template-directed assembly of degenerate oligonucleotides followed by PCR amplification and subcloning. Nucleotide sequence analysis of clones demonstrated that this is an effective method for the simultaneous random mutagenesis of several codons distant within the target gene. This should provide a useful tool for modifying the ligand binding properties of proteins, e.g., the antigen binding affinity and specificity of antibodies.