Site-directed mutagenesis of the Thauera aromatica strain T1 tutE tutFDGH gene cluster

Benzylsuccinate synthase, encoded by the tutF, tutD, and tutG genes of Thauera aromatica strain T1, is responsible for the first step of anaerobic toluene metabolism. Previous work has shown that these genes are part of the tutE tutFDGH gene cluster and strains carrying a mutation in the tutE, tutF, tutD, or tutG genes are unable to metabolize toluene. In this study, we performed site-directed mutagenesis of the tutE, tutF, and tutG genes and determined that the cysteines at position 72 and 79 of TutE are likely to be critical for the radical activation of benzylsuccinate synthase, while the cysteine alanine at positions 9 and 10 of TutF, and the cysteine at position 29 of TutG are also essential for toluene metabolism. Additionally, we report that the tutH gene is necessary for toluene metabolism and the glycine lysine serine (part of the putative ATP/GTP binding domain) at positions 52-54 of the TutH protein is essential for toluene metabolism.     

Role of benzylsuccinate in the induction of the tutE tutFDGH gene complex of T. aromatica strain T1

Expression of the tutE tutFDGH gene cluster of Thauera aromatica strain T1 was examined by Northern and Western analysis in a wild-type strain and chromosomally deleted strains with or without in-frame deletion plasmids. While expression was observed when the wild-type strain was induced with toluene, various chromosomally deleted strains exhibited little or no expression of the tut genes. In contrast, both wild-type and chromosomally deleted strains expressed the tut genes when induced with benzylsuccinate. We conclude that benzylsuccinate is required for the full induction of the tutE tutFDGH gene cluster of T. aromatica strain T1.     

Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.     

Characterization of heme uptake cluster genes in the fish pathogen Vibrio anguillarum

Vibrio anguillarum can utilize hemin and hemoglobin as sole iron sources. In previous work we identified HuvA, the V. anguillarum outer membrane heme receptor by complementation of a heme utilization mutant with a cosmid clone (pML1) isolated from a genomic library of V. anguillarum. In the present study, we describe a gene cluster contained in cosmid pML1, coding for nine potential heme uptake and utilization proteins: HuvA, the heme receptor; HuvZ and HuvX; TonB, ExbB, and ExbD; HuvB, the putative periplasmic binding protein; HuvC, the putative inner membrane permease; and HuvD, the putative ABC transporter ATPase. A V. anguillarum strain with an in-frame chromosomal deletion of the nine-gene cluster was impaired for growth with heme or hemoglobin as the sole iron source. Single-gene in-frame deletions were constructed, demonstrating that each of the huvAZBCD genes are essential for utilization of heme as an iron source in V. anguillarum, whereas huvX is not. When expressed in Escherichia coli hemA (strain EB53), a plasmid carrying the gene for the heme receptor, HuvA, was sufficient to allow the use of heme as the porphyrin source. For utilization of heme as an iron source in E. coli ent (strain 101ESD), the tonB exbBD and huvBCD genes were required in addition to huvA. The V. anguillarum heme uptake cluster shows some differences in gene arrangement when compared to homologous clusters described for other Vibrio species.     

Characterization of the human p57KIP2 gene: Alternative splicing,insertion/deletion polymorphisms in VNTR sequences in the coding region,and mutational analysis

We have isolated human cDNA and genomic clones of a gene termed p57^KIP2, which is related to the p21^WAF1 and p27^KIP1 genes that encode inducible inhibitors of cyclin-dependent kinase activity. The p57 gene contains three GC-rich introns of 166 bp, 566 bp, and 83 bp, and two of the four exons correspond to coding regions. Alternative splicing generates the heterogeneity in the translational initiations. As this gene has been localized to chromosomal band 11p15.5, a region thought to be the location of a tumor suppressor gene(s) for carcinomas of the breast, bladder, and liver, we have examined a large number of tumors for genetic alterations of p57. Although no somatic mutation has been detected, we have found several normal variations in this gene, including four types of 12-bp in-frame deletions in the proline/alanine repeating domain, in which nearly 40 motifs, viz., 5′-CCGGCC-3′, are tandemly repeated. Received: 9 August 1995     

Heterologous expression and identification of the genes involved in anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) in Azoarcus anaerobius

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R(+), carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R(+) carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R(+), eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.     

Characterization of four constant region genes of rabbit immunoglobulin-lambda chains

A cDNA plasmid insert encoding the constant (C) region of a rabbit immunoglobulin-lambda light chain was used as a probe for screening a rabbit liver genomic DNA cosmid library. This allowed the isolation and identification of four distinct C lambda genes, designated C lambda 1, C lambda 2, C lambda 3, and C lambda 4, which were shown to be widely separated from each other along chromosomal DNA. Their nucleotide sequences have been determined. No in-frame termination codons were found within the coding regions. The C lambda 1, C lambda 2, and C lambda 3 sequences are quite similar to each other, but share less homology with the C lambda 4 gene or the cDNA-C lambda sequence used as a probe. The C lambda gene coding for the cDNA sequence was not isolated. Translation of the C lambda 1, C lambda 2, and C lambda 3 sequences predicts a Cys-Pro carboxy-terminal amino acid sequence, as found so far only for horse lambda-chains. Compared to the other rabbit C lambda genes, the C lambda 3 sequence exhibits two deletions, one of 9 bp, the other of 3 bp. The latter occurs at the same position as in the mouse C lambda 2 and C lambda 3 genes. These two deletions are located in the loops between anti-parallel beta-pleated sheets of the C lambda domain. When the C lambda nucleotide sequences from man, mouse, and rabbit are compared, there is less divergence within the same species than for interspecies comparisons. Possible genetic implications of this finding are discussed.     

Close physical linkage of human troponin genes: organization, sequence, and expression of the locus encoding cardiac troponin I and slow skeletal troponin T

Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.     

Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice

The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87–100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants. We also demonstrated proof-of-efficiency of Cas9/sgRNAs in producing large chromosomal deletions (115–245 kb) involving three different clusters of genes in rice protoplasts and verification of deletions of two clusters in regenerated T0 generation plants. Together, these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice and other crops, enabling both basic research and agricultural applications.     

A new mutation delivery system for genome-scale approaches in Bacillus subtilis

In Bacillus subtilis, although many genetic tools have been developed, gene replacement remains labour-intensive and not compatible with large-scale approaches. We have developed a new one-step gene replacement procedure that allows rapid alteration of any gene sequence or multiple gene sequences in B. subtilis without altering the chromosome in any other way. This novel approach relies on the use of upp, which encodes uracil phosphoribosyl-transferase, as a counter-selectable marker. We fused the upp gene to an antibiotic-resistance gene to create an 'upp-cassette'. A polymerase chain reaction (PCR)-generated fragment, consisting of the target gene with the desired mutation joined to the upp-cassette, was integrated into the chromosome by homologous recombination, using positive selection for antibiotic resistance. Then, the eviction of the upp-cassette from the chromosome by recombination between short repeated chromosomal sequences, included in the design of the transforming DNA molecule, was achieved by counter-selection of upp. This procedure was successfully used to deliver a point mutation, to generate in-frame deletions with reduced polar effects, and to combine deletions in three paralogous genes encoding two-component sensor kinases. Also, two chromosome regions carrying previously unrecognized essential functions were identified, and large deletions in two dispensable regions were combined. This work outlines a strategy for identifying essential functions that could be used at genome scale.     

Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization.

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow with toluene as the sole C-source. Tn5 mutagenesis was carried out and a mutant unable to use toluene as the sole C-source was isolated. DNA was sequenced upstream and downstream of the site where the Tn5 was inserted. Analysis of the DNA revealed 13 open reading frames (ORFs) homologous to the tod genes for the toluene dioxygenase pathway of P. putida F1, which are organized in two operons: todXFC1C2BADEGIH and todST. The Tn5 was inserted at the todH gene. The role of the todXFC1C2BADEGIH operon in toluene metabolism was further confirmed in a todC1 mutant (generated by insertional inactivation), which was unable to use toluene as the sole C-source. Primer extension analysis identified a single promoter upstream from the todX gene. The -10 and -35 regions of this promoter showed no significant homology to known promoters. Expression from the todX promoter occurred in response to toluene, ethylbenzene, styrene, xylenes and other aromatic hydrocarbons. Expression from the todS gene was constitutive. Sensitivity to toluene of the todH and todC1 mutants was similar to that of the wild-type strain. This suggests that toluene metabolism is not involved in toluene tolerance.     

The Prevalence of Chromosomal Deletions Relating to Developmental Delay and/or Intellectual Disability in Human Euploid Blastocysts

Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy) and structural (deletion, duplication or others) anomalies. Our previous study indicated that chromosomal deletion(s) is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s) using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34–149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s) are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision.     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50?kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors.