Lys Gene Research Articles

High-lysine proteins in Hiproly barley breeding: Identification, nutritional significance and new screening methods

A 4–7 fold increased content of four major salt-soluble proteins in the barley grain was found to be closely associated with the Hiproly high-lysine character. The proteins were identified by crossed Immunoelectrophoresis as: “free” β-amylase, “free” protein Z and two different chymotrypsin inhibitors, with lysine content of 5.0, 7.1, 9.5, and 11.5 g per 100 g protein, respectively. The two inhibitors seem to be the major high-lysine proteins detected in previous studies of Hiproly. Rocket Immunoelectrophoresis showed that, in total, the four proteins may account for about 7 % of the grain lysine in normal varieties, 17 % in Hiproly lines, and more than 50 % of the increase in grain lysine due to the Hiproly lys gene. Total β-amylase, total protein Z and total chymotrypsin inhibitor seem to contribute equally to this increase. Analyses of Hiproly breeding material showed that screening for the high-lysine character can be based on simple assays for one of the proteins, not depending on determination of total protein in the grain. Especially, the determination of total chymotrypsin inhibitor activity in flour, single-grain or half-grain samples is suggested as a fast and reliable screening test in future breeding with Hiproly.

Improvement of nutritional value in cereals

This investigation is principally aimed at exemplifying the developmental sources (italicized below) that are the requisites to obtain an adequate nutritional role for cereals in feeds and foods. Barley for feed purposes is given central attention and is compared with wheat, rye, ryewheat and oats. The importance of basic research in plant and animal sciences is briefly reviewed, and also complemented with experimental results. Development of reliable, rapid and inexpensive screening methods is stressed and exemplified by a feeding test with mice and a dye-binding (DBC) method for estimating lysine. Screening analyses for quality characters in cereals are used in inventories studying cereals, feed supplements, and complete commercial feeds. The information obtained is tentatively evaluated and is used for promulgating a provisional development programme for Swedish conditions. The research that has been carried out for the realization of the programme is discussed with attention focused particularly on the development of high-lysine barleys and a high-temperature drying process for cereals for livestock feeds. A high-protein, high-lysine barley line (Hiproly) was recovered from the World Barley Collection employing the DBC method. A recessive gene (lys) was found in Hiproly that increases lysine and other essential amino acids in the endosperm independently of protein content. Genetical, plant-physiological, biochemical, ultrastructural and animal nutritional aspects of high-lysine genes in barley and in maize are extensively discussed along with concomitant analyses of the lys gene. Problems involving grain deterioration and its prevention in Scandinavian cereal production are considered. In addition, the effect of heat on the availability of lysine in cereals during storage, drying and baking is experimentally studied. On the basis of the observations and results, a full-scale, rapid, high-temperature drying method has been developed that is adopted in agriculture in Sweden. The results are summarized and confronted with the operational concept of nutritional value. The needs for retrieval of information from research and from the applied sphere of cereal utilization — all for optimal exploitation by plant breeders — are discussed for conditions prevalent in Sweden, on the one hand, and in the developing countries, on the other. Research data from basic sciences as well as information from regional inventories on technological, ecological and social conditions have to be co-ordinated in a synthesis in science specific for each country. New solutions must be transferred to the individual and group level of people who can use them as selection criteria favouring survival and quality in economy and life.

Characterization of a double gene disruption in the LYS2 locus of the pathogenic yeast, Candida albicans

The 4·2 kbp LYS2 gene of Candida albicans encodes the 150 kDa subunit of the 180 kDa heterodimeric enzyme α-aminoadipate reductase. To facilitate structural and functional studies of the LYS2 gene, in this investigation both alleles were sequentially disrupted in C. albicans CAI4. The disruptions were performed using a targeting vector that contained a 2·2 kbp portion of LYS2 from which a 600-bp fragment had been deleted and replaced with a hisG-URA3-hisG cassette. Disruption of both alleles of the LYS2 locus was confirmed by Southern hybridization with appropriate probes. To determine the physiological effect of the LYS2 disruption, cell extracts were assayed for the LYS2-encoded α-aminoadipate reductase activity. The parent, heterozygote and LYS2-disrupted strains exhibited significant, partial and no activity, respectively. Transformation of the LYS2-disrupted strain with the LYS2 gene reversed the mutant phenotype as shown by growth on selective media and restored α-aminoadipate reductase activity. These results demonstrated that the LYS2 gene of C.albicans is responsible for the synthesis of functional α-aminoadipate reductase.

The concentration and yield of hordein and some lysine-rich proteins as influenced by the lys gene of Hiproly barley

Balasaraswathi, R., Køie, B. and Doll, H. 1984. The concentration and yield of hordein and some lysine-rich proteins as influenced by the lys gene of Hiproly barley—Hereditas 100: 225–231. Lund, Sweden. ISSN 0018–0661. Received July 1, 1983 A line having the high-lysine gene lys of ‘Hiproly’ was crossed with a normal variety, and homozygous offspring lines were derived from chromosome-doubled haploids made from the F1. The intluence of the lys gene was tested by comparing the offspring lines having the lys gene with the lines having the corresponding normal gene. The lys lines had 16% lower grain yield and 13% lower single seed weight than the corresponding normal lines. The concentration of hordein in the protein and the yield of this storage protein were strongly reduced in the lys lines. On the contrary, the concentration of protein Z, β-amylase and chymotrypsin inhibitors 1 and 2 was increased 2–6 fold. The yield of these proteins was significantly increased in the lines having the lys gene. However, the absolute increase in the yield of these lysinerich proteins was less than the absolute decrease in hordein yield.

Disintegrator vectors for single‐copy yeast chromosomal integration

Vectors were developed for two-step chromosomal integration of reporter genes or expression constructs. With these vectors, integration produces a disruption of the ADE8, LYS2, MET15, LEU2, HIS3 or FCY1 genes, and integrants can be easily identified by replica-plating on selective media. Integration using these 'disintegrator' vectors produces a single-copy integration of the construct of interest at the junction of the marker deletion, and removes the additional plasmid sequences. Importantly, the integrated constructs do not contain flanking sequence duplications, and therefore should be highly stable. Each of the vectors was shown to reliably integrate a TEF1-KAN expression cassette and/or GAL1-HIS3 and STE12-LacZ reporter genes.

Systems metabolic engineering of Escherichia coli for L ‐threonine production

Amino-acid producers have traditionally been developed by repeated random mutagenesis owing to the difficulty in rationally engineering the complex and highly regulated metabolic network. Here, we report the development of the genetically defined L-threonine overproducing Escherichia coli strain by systems metabolic engineering. Feedback inhibitions of aspartokinase I and III (encoded by thrA and lysC, respectively) and transcriptional attenuation regulations (located in thrL) were removed. Pathways for Thr degradation were removed by deleting tdh and mutating ilvA. The metA and lysA genes were deleted to make more precursors available for Thr biosynthesis. Further target genes to be engineered were identified by transcriptome profiling combined with in silico flux response analysis, and their expression levels were manipulated accordingly. The final engineered E. coli strain was able to produce Thr with a high yield of 0.393 g per gram of glucose, and 82.4 g/l Thr by fed-batch culture. The systems metabolic engineering strategy reported here may be broadly employed for developing genetically defined organisms for the efficient production of various bioproducts.

Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements

Native chromatin IP assays were used to define changes in core histone acetylation at the lysozyme locus during developmental maturation of chicken macrophages and stimulation to high-level expression by lipo-polysaccharide. In pluripotent precursors the lysozyme gene (Lys) is inactive and there is no acetylation of core histones at the gene, its promoter or at the upstream cis-control elements. In myeloblasts, where there is a very low level of Lys expression, H4 acetylation appears at the cis-control elements but not at the Lys gene or its promoter: neither H3 nor H2B become significantly acetylated in myeloblasts. In mature macrophages, Lys expression increases 5-fold: H4, H2B and H2A.Z are all acetylated at the cis-control elements but H3 remains unacetylated except at the −2.4 S silencer. Stimulation with LPS increases Lys expression a further 10-fold: this is accompanied by a rise in H3 acetylation throughout the cis-control elements; H4 and H2B acetylation remain substantial but acetylation at the Lys gene and its promoter remains low. Acetylation is thus concentrated at the cis-control elements, not at the Lys gene or its immediate promoter. H4 acetylation precedes H3 acetylation during development and H3 acetylation is most directly linked to high-level Lys expression.

Altered expression of master regulatory genes of adipogenesis in lipomas from patients bearing tRNA Lys point mutations in mitochondrial DNA

The mechanisms underlying the appearance of lipomas in patients bearing mutations in the tRNA Lys gene of mitochondrial DNA are unknown. We investigated changes in gene expression patterns in lipomas from three patients bearing A8344G or G8363A tRNA Lys gene mutations. Uncoupling protein-1 mRNA was detected in the lipomas, in contrast with undetectable expression in normal adipose tissue. However, expression of other markers of brown fat, such as PGC-1α, was unaltered. PPARγ and retinoblastoma gene expression was down regulated in the lipomas, but C/EBPα mRNA was not affected. The expression of Pref-1 was dramatically down regulated. Thus, lipomatosis due to tRNA Lys mutations is associated with a pattern of altered expression of master regulators of adipogenesis consistent with enhanced proliferation but maintenance of adipocyte features, and with a distorted pattern of brown versus white adipocyte differentiation.

Cloning the Schizosaccharomyces pombe lys2 + gene and construction of new molecular genetic tools

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2(+) gene, which is homologous to Saccharomyces cerevisiae LYS4(+). We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2(+)-based cloning vector pRH3. In addition, we deleted the S. pombe his7(+) gene with a lys2(+) -marked polymerase chain reaction (PCR) product and the S. pombe lys2(+) gene with a his7(+)-marked PCR product. Strains carrying these deletions of lys2(+) or his7(+) serve as relatively efficient hosts for the deletion of the ade6(+) gene by lys2(+)-- or his7(+)--marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2(+) as a selectable marker, along with improved strains for the use of his7(+) to mark gene deletions.

Coordinate regulation of multiple and distinct biosynthetic pathways by TOR and PKA kinases in S. cerevisiae

The target of rapamycin (TOR) signaling pathway is an essential regulator of cell growth in eukaryotic cells. In Saccharomyces cerevisiae, TOR controls the expression of many genes involved in a wide array of distinct nutrient-responsive metabolic pathways. By exploring the TOR pathway under different growth conditions, we have identified novel TOR-regulated genes, including genes required for branched-chain amino acid biosynthesis as well as lysine biosynthesis (LYS genes). We show that TOR-dependent control of LYS gene expression occurs independently from previously identified LYS gene regulators and is instead coupled to cAMP-regulated protein kinase A (PKA). Additional genome-wide expression analyses reveal that TOR and PKA coregulate LYS gene expression in a pattern that is remarkably similar to genes within the ribosomal protein and "Ribi" regulon genes required for ribosome biogenesis. Moreover, this pattern of coregulation is distinct from other clusters of TOR/PKA coregulated genes, which includes genes involved in fermentation as well as aerobic respiration, suggesting that control of gene expression by TOR and PKA involves multiple modes of crosstalk. Our results underscore how multiple signaling pathways, general growth conditions, as well as the availability of specific nutrients contribute to the maintenance of appropriate patterns of gene activity in yeast.

Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap

Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MS(n) spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the "lock mass" by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.

RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples.

We have developed RNA expression microarrays (REMs), in which each spot on a glass support is composed of a population of cDNAs synthesized from a cell or tissue sample. We used simultaneous hybridization with test and reference (housekeeping) genes to calculate an expression ratio based on normalization with the endogenous reference gene. A test REM containing artificial mixtures of liver cDNA and dilutions of the bacterial LysA gene cDNA demonstrated the feasibility of detecting transcripts at a sensitivity of four copies of LysA mRNA per liver cell equivalent. Furthermore, LysA cDNA detection varied linearly across a standard curve that matched the sensitivity of quantitative real-time PCR. In REMs with real samples, we detected organ-specific expression of albumin, Hnf-4 and Igfbp-1, in a set of mouse organ cDNA populations and c-Myc expression in tumor samples in paired tumor/normal tissue cDNA samples. REMs extend the use of classic microarrays in that a single REM can contain cDNAs from hundreds to thousands of cell or tissue samples each representing a specific physiological or pathophysiological state. REMs will extend the analysis of valuable samples by providing a common broad based platform for their analysis and will promote research aimed at defining gene functions, by broadening our understanding of their expression patterns in health and disease.

HIV disease progression and limited antiretroviral treatment options for a HIV-1 infected individual with myoclonic epilepsy associated with ragged red fibers

We describe a 50-year-old Caucasian man with a family history of myoclonic epilepsy associated with ragged red fibers (MERRF) and a diagnosis of Human Immunodeficiency Virus (HIV). The patient had multiple risk factors for contracting HIV and was being followed in our clinic at the time of his diagnosis. Initial testing following seroconversion revealed a baseline CD4+ T-lymphocyte count of 652×10 6 cells/l and a HIV-1 RNA of 14,781 copies/ml. He reported exercise intolerance and had mild neurologic deficits, which worsened around the time of HIV seroconversion. These symptoms led to his subsequent diagnosis of MERRF by the detection of the A8344G point mutation in the tRNA Lys gene of mitochondrial DNA (mtDNA). The baseline estimated proportion of mutant genome was 39%. He showed a rapid course of HIV disease progression with a CD4+ T-lymphocyte nadir of 174×10 6 cells/l associated with a HIV-1 RNA of 238,178 copies/ml, within 17 months following HIV seroconversion. To avoid further mitochondrial insult, which could result from the use of a standard nucleoside reverse transcriptase inhibitor-containing regimen, a protease inhibitor regimen consisting of hard-gel saquinavir (Invirase™), and lopinavir/ritonavir (Kaletra™) was chosen for this patient. The patient's CD4+ T-lymphocyte count increased to 282×10 6 cells/l and his viral load became undetectable 7 months following the initiation of antiretroviral therapy. His neurologic symptoms did not worsen on this antiretroviral regimen. When initiating HIV therapy in individuals with metabolic myopathies related to mitochondrial dysfunction, it may be important to design an antiviral regimen that minimizes mitochondrial damage, yet effectively maintains durable viral suppression.

Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303.

phi-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of phi-0303 in Escherichia coli DH5alpha. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of phi-0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCR amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages. Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium.

Generation of an Escherichia coli lysA targeted deletion mutant by double cross-over recombination for potential use in a bacterial growth-based lysine assay.

To generate a stable Escherichia coli lysine auxotroph for the lysine bioavailability assay. An E. coli lysine auxotrophic strain was constructed by deleting the entire lysA gene and replacing it with a gene that confers resistance to ampicillin (bla). The linear DNA contained 50 bp homologous sequence of upstream of lysA in one end and 50 bp of downstream of lysA in the other end. The deltalysA::bla strain exhibited a linear response to lysine supplementation and can be used for quantifying lysine.

Functionalized self-assembled monolayer on gold for detection of human mitochondrial tRNA gene mutations

We developed a rapid and simple method to identify single-nucleotide polymorphisms (SNPs) in the human mitochondrial tRNA genes. This method is based on a universal, functionalized, self-assembled monolayer, XNA on Gold chip platform. A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was immobilized on chips and the chips were then hybridized with fluorescence-labeled reference targets produced by asymmetric polymerase chain reaction from patient DNA. The ratio of the hybridization signals from the reference and test targets with each probe was then calculated. A ratio of above 3 indicates the presence of a wild-type sequence and a ratio of below 0.3 indicates a mutant sequence. We tested the sensitivity of the chip for known mutations in tRNA Leu(UUR) and tRNA Lys genes and found that it can also be used to discriminate multiple mutations and heteroplasmy, two typical features of human mitochondrial DNA. The XNA on Gold biochip method is a simple and rapid microarray method that can be used to test rapidly and reliably any SNP in the mitochondrial genome or elsewhere. It will be particularly useful for detecting SNPs associated with human diseases.

Molecular characterization of the Candida albicans LYS5 gene and site-directed mutational analysis of the PPTase (Lys5p) domains for lysine biosynthesis

The LYS2 and LYS5 genes of the pathogenic yeast Candida albicans are required for the α-aminoadipate reductase (AAR) reaction in the lysine biosynthetic pathway. The LYS2 encodes an apo-AAR (Lys2p) and the LYS5 encodes a phosphopantetheinyl transferase (PPTase) for the post-translational activation of AAR. Our cloned C. albicans LYS5 gene encodes a 38.4 kDa PPTase which is 27% identical and 43% similar to the Saccharomyces cerevisiae Lys5p. Sequence alignment of Lys5p with other PPTases reveals highly conserved putative PPTase domains including the Core 3, WXXKESXXK (residues 194–202). Recombinant Lys5p expressed in Escherichia coli activates C. albicans Lys2p for the AAR activity and also activates AARs from S. cerevisiae and to a lesser extent Schizosaccharomyces pombe. Site-directed mutational analyses reveal glutamic acid 198 in the Lys5p Core 3 as essential for the activation of recombinant Lys2p AAR activity. Other conserved amino acids were also analyzed for their influence on Lys5p PPTase activity. Our results demonstrate cloning of the LYS5 gene, expression of Lys5p, in vitro Lys2p activation model and characterization of the functional motifs of the C. albicans PPTase.

Characterization of an Escherichia coli lysA insertion targeted mutant using phenotype arrays

The objective of this study was to investigate the effect of a lysine biosynthesis insertion mutation on the growth response and phenotype of Escherichia coli. The lysA gene encodes the last enzyme in the lysine biosynthetic pathway in most bacteria. This E. coli insertion mutant exhibited altered growth physiology and phenotype of the recipient E. coli. The constructed mutant could grow in the absence of lysine supplementation although the extent of growth after 7 h incubation in the presence of most lysine concentration was significantly ( p<0.05) decreased compared to that observed with the parent E. coli strain. The mutant was also less able to utilize carbon and nitrogen substrates than the parent E. coli strain as determined by using phenotype arrays. These results suggest that the carbon and nitrogen phenotype profiles of E. coli when measured on phenotype arrays are altered after targeted insertion mutagenesis in the lysA gene. Creation of altered phenotypes may have potential for pharmaceutical and biotechnological applications of lysine E. coli metabolism.

Crystal Structure of Mycobacterium tuberculosis Diaminopimelate Decarboxylase, an Essential Enzyme in Bacterial Lysine Biosynthesis

The Mycobacterium tuberculosis lysA gene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5'-phosphate (PLP)-dependent enzyme. The enzyme catalyzes the final step in the lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) to l-lysine. The lysA gene of M. tuberculosis H37Rv has been established as essential for bacterial survival in immunocompromised mice, demonstrating that de novo biosynthesis of lysine is essential for in vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and the three-dimensional structure of DAPDC from M. tuberculosis complexed with reaction product lysine and the ternary complex with PLP and lysine in the active site has been determined. The first structure of a DAPDC confirms its classification as a fold type III PLP-dependent enzyme. The structure shows a stable 2-fold dimer in head-to-tail arrangement of a triose-phosphate isomerase (TIM) barrel-like alpha/beta domain and a C-terminal beta sheet domain, similar to the ornithine decarboxylase (ODC) fold family. PLP is covalently bound via an internal aldimine, and residues from both domains and both subunits contribute to the binding pocket. Comparison of the structure with eukaryotic ODCs, in particular with a di-fluoromethyl ornithine (DMFO)-bound ODC from Trypanosoma bruceii, indicates that corresponding DAP-analogues might be potential inhibitors for mycobacterial DAPDCs.

Impact of the cross-pathway control on the regulation of lysine and penicillin biosynthesis in Aspergillus nidulans.

The non-proteinogenic amino acid, alpha-aminoadipate, defines the biosynthetic branch-point of lysine and penicillin biosynthesis in the filamentous fungus, Aspergillus nidulans. Regulation of both pathways was analysed in response to amino acid limitation. The lysF-encoded homoaconitase acts upstream of the alpha-aminoadipate branch point, whereas the lysA gene product, saccharopine dehydrogenase, catalyses the ultimate step of the lysine-specific branch. The lysA gene from A. nidulans was identified and isolated. Amino acid starvation resulted in significantly increased transcription of lysA but not lysF. Starvation-dependent changes in transcription levels of lysA were dependent on the presence of the central transcriptional activator of the cross-pathway control (CPCA). The effect of amino acid starvation under penicillin-producing conditions was analysed in A. nidulans strains with reporter genes for the penicillin-biosynthesis genes, acvA and ipnA, and genetically altered activity of the cross-pathway control. Overproduction of CPCA decreased expression of ipnAand acvA reporter genes and even more drastically reduced penicillin production. This work suggests that, upon amino acid starvation, the cross-pathway control overrules secondary metabolite biosynthesis and favours the metabolic flux towards amino acids instead of penicillin in A. nidulans.