Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs.

A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the BglII recognition sequence (AGATCT) in the GFPUV gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.     

Random tag insertions by Transposon Integration mediated Mutagenesis (TIM)

Transposon Integration mediated Mutagenesis (TIM) is a broadly applicable tool for protein engineering. This method combines random integration of modified bacteriophage Mu transposons with their subsequent defined excision employing type IIS restriction endonuclease AarI. TIM enables deletion or insertion of an arbitrary number of bases at random positions, insertion of functional sequence tags at random positions, replacing randomly selected triplets by a specific codon (e.g. scanning) and site-saturation mutagenesis. As a proof of concept a transposon named GeneOpenerAarIKan was designed and employed to introduce 6xHis tags randomly into the esterase EstC from Burkholderia gladioli. A TIM library was screened with colony based assays for clones with an integrated 6xHis tag and for clones exhibiting esterase activity. The employed strategy enables the isolation of randomly tagged active enzymes in single mutagenesis experiments.     

Murine sarcoma virus ts110 RNA transcripts: origin from a single proviral DNA and sequence of the gag-mos junctions in both the precursor and spliced viral RNAs

Our previous studies have argued persuasively that in murine sarcoma virus ts110 (MuSVts110) the gag and mos genes are fused out of frame due to a approximately 1.5-kilobase (kb) deletion of wild-type murine sarcoma virus 349 (MuSV-349) viral information. As a consequence of this deletion, infected cells grown at 39 degrees C appear morphologically normal, producing a 4-kb viral RNA and a truncated gag gene product, P58gag. At 33 degrees C, however, MuSVts110-infected cells appear transformed, producing two viral RNAs, about 4 and 3.5 kb in length, and two viral proteins, P58gag and P85gag-mos. Recent S1 nuclease analyses (Nash et al., J. Virol. 50:478-488, 1984) suggested strongly that at 33 degrees C about 430 bases surrounding the out-of-frame gag-mos junction and bounded by consensus splice donor and acceptor sites are excised from the 4-kb RNA to form the 3.5-kb RNA. As a result of this apparent splicing event, the gag and mos genes seemed to be fused in frame and allowed the translation of P85gag-mos. In the present study, DNA primers hybridizing to the MuSVts110 4- and 3.5-kb RNAs just downstream of the gag-mos junction points were used to sequence these junctions by the primer extension method. We observed that, relative to wild-type MuSV-349 5.2-kb RNA, the MuSVts110 4-kb RNA had suffered a 1,488-base deletion as a result of the fusion of wild-type gag gene nucleotide 2404 to wild-type mos gene nucleotide 3892. This gag-mos junction is out of frame, containing both TAG and TGA termination codons in the reading frame 42 and 50 bases downstream of the gag-mos junction, respectively. Thus, the MuSVts110 4-kb RNA can only be translated into a truncated gag precursor containing an additional C-terminal 14 amino acid residues derived from an alternate mos gene reading frame. Similar analyses of the MuSVts110 3.5-kb RNA showed a further loss of both gag and mos sequences over those deleted in the original 1,488-base deletion. In the MuSVts110 3.5-kb RNA, we found that gag nucleotide 2017 was fused to mos nucleotide 3936 (nucleotide 2449 in the MuSVts110 4-kb genome). This 431-base excised fragment is bounded exactly by in-frame consensus splice donor and acceptor sequences. As a consequence of this splice event, the TAG codon is excised and the restoration of the original mos gene reading frame allows the TGA codon to be bypassed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alteration of the Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader by mitochondrial gene replacement and functional interaction with the translational activator protein PET111.

The ability to replace wild-type mitochondrial DNA sequences in yeast with in vitro-generated mutations has been exploited to study the mechanism by which the nuclearly encoded PET111 protein specifically activates translation of the mitochondrially coded COX2 mRNA. We have generated three mutations in vitro that alter the COX2 mRNA 5'-untranslated leader (UTL) and introduced them into the mitochondrial genome, replacing the wild-type sequence. None of the mutations significantly affected the steady-state level of COX2 mRNA. Deletion of a single base at position -24 (relative to the translation initiation codon) in the 5'-UTL (cox2-11) reduced COX2 mRNA translation and respiratory growth, whereas insertion of four bases in place of the deleted base (cox2-12) and deletion of bases -30 to -2 (cox2-13) completely blocked both. Six spontaneous nuclear mutations were selected as suppressors of the single-base 5'-UTL deletion, cox2-11. One of these mapped to PET111 and was shown to be a missense mutation that changed residue 652 from Ala to Thr. This suppressor, PET111-20, failed to suppress the 29-base deletion, cox2-13, but very weakly suppressed the insertion mutation, cox2-12. PET111-20 also enhanced translation of a partially functional COX2 mRNA with a wild-type 5'-UTL but a mutant initiation codon. Although overexpression of the wild-type PET111 protein caused weak suppression of the single-base deletion, cox2-11, the PET111-20 suppressor mutation did not function simply by increasing the level of the protein. These results demonstrate an intimate functional interaction between the translational activator protein and the mRNA 5'-UTL and suggest that they may interact directly.     

Deletion and insertion mutations in early region 1a of type 5 adenovirus that produce cold-sensitive or defective phenotypes for transformation

On the basis of earlier findings showing that H5hr1 (hr1) is cold sensitive for transformation, a series of mutants were constructed so that they contained deletions or insertions in different sites of early region 1a (E1a) to ascertain: (i) whether the cold-sensitive phenotype of hr1 was the result of the identified single-base pair deletion of nucleotide 1,055 or due to a missense mutation at another site and (ii) what region and how much of the E1a 51-kilodalton protein is actually required to produce cell transformation. A mutant, H5dl101 (dl101), was constructed to contain a 5-base pair deletion of nucleotides 1,008 to 1,012, which produced a frameshift and a subsequent stop codon at nucleotide 1,241. This mutant, which should encode a truncated 33-kilodalton protein in place of the wild-type 51-kilodalton protein, had a cold-sensitive phenotype for transformation essentially identical to hr1. Consonant with this finding, a mutant (H5in106) engineered to contain a 16-base pair insertion initiated after nucleotide 1,009, with a stop codon beginning at the newly inserted nucleotide 1,013, also had a cold-sensitive phenotype like hr1 and dl101. It is striking, however, that a mutant (H5dl105) with a 69-base pair deletion beginning at nucleotide 1,003, and having a stop codon at nucleotide 1,544, was totally defective for transformation at any temperature. Transfection studies with plasmids containing the E1a or E1a and E1b genes of sub309, hr1, and dl101 further revealed that the cold-sensitive transformation phenotype observed could be exhibited in the absence of viral E1b gene expression.     

Characterization of the translational start site for IF2 beta, a short form of Escherichia coli initiation factor IF2

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.     

Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101

A plasmid pAPP1 with a 4 kbp insert at the PstI site of pBR322, encoding aminopeptidase P gene of Escherichia coli HB101 (Yoshimoto et al. (1988) J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37 degrees C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of the enzyme was estimated to be 49,650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn2+.     

DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis.

An in vivo assay was used to define the DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis (G4 origin). This assay made use of an origin-cloning vector, mRZ1000, a defective M13 recombinant phage deleted for its natural origin of complementary-strand DNA synthesis. The minimal DNA sequence of the G4 genome sufficient for the restoration of normal M13 growth parameters was determined to be 139 bases long, located between positions 3868 and 4007. This G4-M13 construct was also found to give rise to proper initiation of complementary-strand synthesis in vitro. The cloned DNA sequence contains all the regions of potential secondary structure which have been implicated in primase-dependent replication initiation as well as additional sequence information. To address the role of one region which potentially forms a DNA secondary structure, the DNA sequence internal to the G4 origin was altered by site-directed mutagenesis. A 3-base insertion at the AvaII site as well as a 17-base deletion between the AvaI and AvaII sites both resulted in loss of origin function. The 17-base deletion was also generated within the G4 genome and found to dramatically reduce the infectious growth rate of the resulting phage. These results are discussed with respect to the role of the G4 origin as the recognition site for primase-dependent replication initiation and its possible role in stage II replication.     

A novel complex mutation in the LDL receptor gene probably caused by the simultaneous occurrence of deletion and insertion in the same region

A novel complex mutation with the presence of both deletion and insertion in very close proximity in the same region was detected in exon 8 of the LDL receptor gene from two apparently unrelated Japanese families with familial hypercholesterolemia (FH). In this mutant LDL receptor gene, the nine bases from nucleotide (nt) 1115 to nt 1123 (AGGGTGGCT) were replaced by six different bases (CACTGA), and consequently the four amino acids from codon 351 to 354, Glu-Gly-Gly-Tyr, were replaced by three amino acids, Ala-Leu-Asn, in the conserved amino acid region of the growth factor repeat B of the LDL receptor. The nature of the amino acid substitution and data on the families suggest that this mutation is very likely to affect the LDL receptor function and cause FH. The generation of this complex mutation can be explained by the simultaneous occurrence of deletion and insertion through the formation of a hairpin-loop structure mediated by inverted repeat sequences. Thus this mutation supports the hypothesis that inverted repeat sequences influence the stability of a given gene and promote human gene mutations.     

Increased expression of hepatitis B virus surface antigen gene in Escherichia coli by IS1 insertion

Summary We selected faster growing colonies of Escherichia coli harbouring an expression plasmid for hepatitis B virus surface antigen (HBsAg) gene after mutagenesis. Among these colonies, three were found to produce an increased level of HBsAg as a consequence of alteration of the plasmid. Analysis of this plasmid showed that an insertion sequence, IS1, was inserted into a middle region of the HBsAg gene (codon for Pro 127) to generate a termination codon 20 bp downstream from the junction site between the HBsAg gene and the left end of IS1. Insertion of a chemically synthesized termination codon into the same region of the HBsAg gene also increased the expression of the HBsAg gene. These results suggest that HBsAg lacking the COOH-terminal region is produced at a high level because it does not inhibit the growth of the host.     

Segment IV of a Salmonella flagellin gene specifies flagellar antigen epitopes

Each of the two mutants isolated from a fliC (= hag, flagellin-deficient) Escherichia coli strain made motile by a plasmid carrying the fliC gene of Salmonella muenchen by selection for motility in the presence of anti-d (Salmonella flagellar antigen) serum had both lost and gained one or more subfactors of the wild-type antigen. In one mutant codon 246 was GAC (alanine) instead of GCC (asparagine); the other had a deletion of 105 base pairs, explicable by a 10bp direct repeat, starting at bases 782 and 887. The in vitro removal of a 48bp EcoRV(631)/EcoRV(679) fragment produced plasmid pLS408, which was found to lack a subfactor of wild-type antigen d but able to confer motility on flagellin-negative Salmonella sp. (and used for insertion of epitope-specifying oligonucleotides at its EcoRV site). Immunoblotting with absorbed and unabsorbed sera from rabbits immunized with E. coli with wild-type or mutated antigen d showed that the fusion proteins specified by lambda gt11 with the N-terminal part of gene lacZ joined to a restriction fragment coding for residues 145-391 of flagellin gave the same pattern of parent-specific and mutant-specific reactions as the flagellate bacteria. Four out of five similarly selected mutants had the same 105 bp deletion as the first-isolated mutant; the fifth had a 72 bp deletion made possible by a 7-base pair direct repeat, starting at positions 649 and 721. All these changes in serological character without loss of function affected segment IV, specifying residues 182 to 308 of the total of 505, where there is little homology between different flagellar-antigen alleles.     

Random mutagenesis of the proton-coupled folate transporter (SLC46A1), clustering of mutations, and the bases for associated losses of function

Loss-of-function mutations in the proton-coupled folate transporter (PCFT, SLC46A1) result in the autosomal recessive disorder, hereditary folate malabsorption (HFM). Identification and characterization of HFM mutations provide a wealth of information on the structure-function relationship of this transporter. In the current study, PCR-based random mutagenesis was employed to generate unbiased loss-of-function mutations of PCFT, simulating the spectrum of alterations that might occur in the human disorder. A total of 26 mutations were generated and 4 were identical to HFM mutations. Eleven were base deletion or insertion mutations that led to a frameshift and, along with similar HFM mutations, are predominantly localized to two narrow regions of the pcft gene at the 5'-end. Base substitution mutations identified in the current study and HFM patients were largely distributed across the pcft gene. Elimination of the ATG initiation codon by a one-base substitution (G > A) did not result in a complete lack of translation at the same codon consistent with rare non-ATG translation initiation. Among six missense mutants evaluated, three mutant PCFTs were not detected at the plasma membrane, one mutation resulted in decreased binding to folate substrate, and one had a reduced rate of conformational change associated with substrate translocation. The remaining PCFT mutant had defects in both processes. These results broaden understanding of the regions of the pcft gene prone to base insertion and deletion and inform further approaches to the analysis of the structure-function of PCFT.     

Enhancement of translation initiation by A/T-rich sequences downstream of the initiation codon in Escherichia coli

The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher beta-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression.     

Integrons found in different locations have identical 5' ends but variable 3' ends.

The positions of the outer boundaries of the 5'- and 3'-conserved segment sequences of integrons found at several different locations have been determined. The position of the 5' end of the 5'-conserved segment is the same for six independently located integrons, In1 (R46), In2 (Tn21), In3 (R388), In4 (Tn1696), In5 (pSCH884), and In0 (pVS1). However, the extent of the 3'-conserved segment differs in each integron. The sequences of In2 and In0 diverge first from the conserved sequence, and their divergence point corresponds to the 3'-conserved segment endpoint defined previously (H.W. Stokes and R.M. Hall, Mol. Microbiol. 3:1669-1683, 1989), which now represents the endpoint of a 359-base deletion in In0 and In2. The sequence identity in In3, In1, In4, and In5 extends beyond this point, but each sequence diverges from the conserved sequence at a different point within a short region. Insertions of IS6100 were identified adjacent to the end of the conserved region in In1 and 123 bases beyond the divergence point of In4. These 123 bases are identical to the sequence found at the mer end of the 11.2-kb insertion in Tn21 but are inverted. In5 and In0 are bounded by the same 25-base inverted repeat that bounds the 11.2-kb insert in Tn21, and this insert now corresponds to In2. However, while In0, In2, and In5 have features characteristic of transposable elements, differences in the structures of these three integrons and the absence of evidence of mobility currently preclude the identification of all of the sequences associated with a functional transposon of this type.     

Alternative splicing of testis-specific lactate dehydrogenase C gene in mammals and pigeon

The objective of the present study was to confirm the widespread existence of alternative splicing of lactate dehydrogenase c (ldhc) gene in mammals. RT-PCR was employed to amplify cDNAs of ldhc from testes of mammals including pig, dog, rabbit, cat, rat, and mouse, as well as pigeon. Two to six kinds of splice variants of ldhc were observed in the seven species as a result of deletion of one or more exons or insertion of partial sequence of an intron in the mature mRNA. The deleted exons occur mostly in exons 5, 4, 6, and 3. The insertion of a partial sequence of introns, which resulted in an abnormal stop codon in the inserted intron sequence, was observed only in dog and rat. The deletion of exons also resulted in a reading frame shift and formation of a stop codon in some variants. No alternative splicing was observed for ldha and ldhb genes in testis of yak. Native polyacrylamide gel electrophoresis and Western blot analysis revealed no obvious LDH-C4 activity derived from expressed ldhc variants. Our results demonstrated the widespread and unique existence of alternative splicing of ldhc genes in mammals.