Various short autonomously replicating sequences from the yeast Kluyveromyces marxianus seemingly without canonical consensus.

Eukaryotic autonomously replicating sequences (ARSs) are composed of three domains, A, B, and C. Domain A is comprised of an ARS consensus sequence (ACS), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. In Saccharomyces cerevisiae and Kluyveromyces lactis ARS101, the ACS is commonly composed of 11 bp, 5'-(A/T)AAA(C/T)ATAAA(A/T)-3'. This core sequence is essential for S. cerevisiae and K. lactis ARS activity. In this study, we identified ARS-containing sequences from genomic libraries of the yeast Kluyveromyces marxianus DMKU3-1042 and validated their replication activities. The identified K. marxianus DMKU3-1042 ARSs (KmARSs) have very effective replication ability but their sequences are divergent and share no common consensus. We have carried out point mutations, deletions, and base pairs substitutions within the sequences of some of the KmARSs to identify the sequence(s) that influence the replication activity. Consensus sequences same as the 11 bp ACS of S. cerevisiae and K. lactis were not found in all minimum functional KmARSs reported here except KmARS7. Moreover, partial sequences from different KmARSs are interchangeable among each other to retain the ARS activity. We have also specifically identified the essential nucleotides, which are indispensable for replication, within some of the KmARSs. Our deletions analysis revealed that only 21 bp in KmARS18 could retain the ARS activity. The identified KmARSs in this study are unique compared to other yeasts' ARSs, do not share common ACS, and are interchangeable.

Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date.

High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast?

The process of ethanol fermentation has a long history in the production of alcoholic drinks, but much larger scale production of ethanol is now required to enable its use as a substituent of gasoline fuels at 3%, 10%, or 85% (referred to as E3, E10, and E85, respectively). Compared with fossil fuels, the production costs are a major issue for the production of fuel ethanol. There are a number of possible approaches to delivering cost-effective fuel ethanol production from different biomass sources, but we focus in our current report on high-temperature fermentation using a newly isolated thermotolerant strain of the yeast Kluyveromyces marxianus. We demonstrate that a 5 degrees C increase only in the fermentation temperature can greatly affect the fuel ethanol production costs. We contend that this approach may also be applicable to the other microbial fermentations systems and propose that thermotolerant mesophilic microorganisms have considerable potential for the development of future fermentation technologies.

Random and targeted gene integrations through the control of non-homologous end joining in the yeast Kluyveromyces marxianus.

Kluyveromyces marxianus DMKU3-1042 is a thermotolerant yeast strain suitable for high-temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 x 10(6) transformants/microg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3-1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non-homologous end-joining (NHEJ) pathway. In yeast lacking KU70, high-frequency non-homologous gene integration was abolished and the Kmku70 mutants showed 82-95% homologous gene targeting efficiencies using homologous sequences of 40-1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid-free gene manipulations in K. marxianus.

Construction of flocculent Kluyveromyces marxianus strains suitable for high-temperature ethanol fermentation.

Flocculating yeasts are highly useful in fermentation processes because these cells can be separated easily from the fermentation mash. However, native yeasts are usually non-flocculating, including Kluyveromyces marxianus, which exhibits a potent high-temperature ethanol fermentation ability. We describe here the construction of flocculent K. marxianus strains via the introduction of the FLO1, FLO5, FLO9, and FLO10 genes from Saccharomyces cerevisiae. The S. cerevisiae FLO genes were overexpressed by upstream insertion of the constitutive TDH3 promoter, resulting in flocculent S. cerevisiae strains. These TDH3p-FLO sequences were then amplified by PCR and introduced directly into a K. marxianus strain. These K. marxianus strains showed a flocculation phenotype, indicating that the introduced S. cerevisiae TDH3 promoter and all FLO genes were functional in this strain. Moreover, a flocculating K. marxianus strain showed the same ethanol production profile as that of its wild-type parent. The K. marxianus flocculating strains we generated should be useful in the future development of cost-effective fed-batch and continuous fermentation systems at high temperatures.

Frequency of intestinal parasites among food-handlers in Khartoum, Sudan.

Food-handlers (n = 1500) attending the public health laboratory in Khartoum, Sudan, for annual check-ups were screened for intestinal parasites by 3 different techniques (direct faecal examination, formol-ether concentration and floatation) to evaluate the adequacy of annual screening. Results showed that 29.4% of food-handlers were harbouring intestinal protozoa in stool samples: Entamoeba coli in 15.3%, Giardia lamblia in 9.7%, and Enta. histolytica in 4.3%. Moreover, 2.7% of food-handlers harboured intestinal helminths: Hymenolepis nana (1.6%), Schistosoma mansoni (0.7%), Taenia saginata (0.3%) and Strongyloides stercoralis (0.1%). We recommend more frequent screening of food-handlers and that the direct faecal smear technique is efficient for the detection of such parasites.

High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast Kluyveromyces marxianus DMKU3-1042.

We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45 degrees C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura(+) transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys(-) transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus alpha-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus alpha-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools.

Economic cost of malaria on households during a transmission season in Khartoum State, Sudan.

This study was conducted in 2004 among 1200 households in Khartoum to estimate the direct and indirect economic costs of malaria for households. Information on the household and the malaria episodes was collected (care-seeking behaviour, working days lost and expenditure on malaria treatment). There were 327 episodes of malaria; 25.2% of the households reported at least 1 malaria episode during the month preceding the survey. In only 18.0% of malaria episodes was the individual economically active. The average treatment expenditure per fully cured case was US$ 6.3 (SD 5.9). The average indirect cost per fully cured case was U.S. $3.2 (SD 9.2); it was higher for individuals working in the informal sector than those employed in the formal sector.

Analysis of hydrolytic activity of a 65-kDa chitinase from the silkworm, Bombyx mori.

The hydrolytic reactions of Bombyx mori 65-kDa chitinase with the short substrates, N-acetyl-chitooligosaccharides, were analyzed by HPLC. Analysis of the hydrolyzed products showed that the newly produced oligosaccharides are all beta anomers, suggesting that, similar to other family 18 glycosyl hydrolases, the 65-kDa chitinase acts in the retaining mechanism. Furthermore, the enzyme cleaves the N-acetylchitooligosaccharides mainly at the linkage between the second and the third GlcNAc moieties from the non-reducing end, while the other sites were cleaved in smaller proportions. Moreover, the initial reaction rates of the enzyme with the longer N-acetylchitooligosaccharides were higher than those with shorter ones. These results suggest that the enzyme is an endo-cleaving type and more efficient on the longer substrates.

Alternative splicing of the primary transcript generates heterogeneity within the products of the gene for Bombyx mori chitinase.

The gene of chitinase in the silkworm, Bombyx mori, generates four mRNA products by alternative splicing. Nucleotide sequences of the entire gene for chitinase and respective cDNAs demonstrate that the pre-mRNA undergoes alternative splicing at both the 5' and 3' regions. At the 5' region, the pre-mRNA experienced differential splicing through two alternative 5'-intron consensus splicing sites. These products differ in the last amino acid of the signal peptide and the first amino acid of the mature N-terminal sequences: one with Cys(20)-Ala(21) and the other with Ser(20)-Asp(21). The product with Cys(20)-Ala(21) residues is one amino acid larger than the other with Ser(20)-Asp(21). At the 3' region the pre-mRNA of the chitinase gene undergoes alternative splicing in three different fashions. It is spliced either through retaining or excluding the upstream 121-bp direct repeat found at the 3' region of the coding sequences or through retaining or excluding of an insertion of 9 bp in a combinatorial manner. Retention or exclusion of the upstream 121-bp direct repeat results in a protein with a deduced amino acid sequence similar in size to the one retaining both direct repeats. However, exclusion of the insert of the 9 bp from the mRNA results in a protein with 22 extra amino acids. All of the mRNA products appear to be generated from a single gene as demonstrated by testing the 3' region of the genomic DNA and variant chitinase mRNA products. B. mori chitinase expression in the fifth instar larvae epidermal tissues appears to be developmentally regulated, but the phenomenon of alternative splicing of the pre-mRNA is not stage-dependent. Furthermore, the four mRNA products showed chitinase activity when expressed in Escherichia coli, which demonstrates the role of the alternative splicing process in generating multiple isoforms of the silkworm's chitinase.

Prevalence of iron deficiency in Saudi children from birth to 15 months of age.

A cross-sectional study was carried out to determine the prevalence of iron deficiency among healthy Saudi children from birth to 15 months of age. The groups studied were: newborns, 3-4 months, 5-6 months, 7-8 months, 9-10 months and 12-15 months of age. The age groups were dictated by the vaccination schedule. Serum ferritin was measured and transferrin saturation calculated in each subject. The lower limits of normal were taken as a transferrin saturation of less than 10% and a serum ferritin of less than 12 micrograms/l. A total of 333 serum samples was adequate for analysis. None of the newborns or the 3-4-month-old infants had evidence of iron deficiency. At 5-6 months only 3.3% of subjects had iron deficiency. In the subsequent older age groups the prevalence of iron deficiency increased significantly with age from 9.3% to 12.7% and reached 14.5% in the oldest age group. Screening for iron deficiency in children attending well-baby clinics and hospitals at ages of 12-15 months is recommended.

The pattern of infection of the nervous system in Riyadh: a review of 121 cases.

Analysis of 121 consecutive cases with infection of the nervous system showed that the majority were the granulomatous infections, tuberculosis and brucellosis (53 cases (44 per cent)). Thirty-nine patients had tuberculosis and 14 had brucellosis. The clinical and microbiological pattern of infection differs from that frequently reported from Western countries. Tuberculosis lesions presented with features of intracranial space occupying lesions (14), spinal cord compression (13) and lumbosacral root compression (1 child). Ten adults and one child had tuberculous meningitis. Pyogenic meningitis present in 38 cases (31 per cent), was most common in children. The infecting organism was identified in 26 patients; Gram-positive cocci in 17, Haemophilus influenzae in four and other Gram-negative organisms in five. Eleven patients had brain abscesses, caused by bacterial infection in eight, fungal infection in two and Toxoplasma gondii in one. Nineteen patients had clinical and pathological features of viral meningitis. Fourteen patients (12 per cent) died including six children with pyogenic meningitis.

Comparison between serum ferritin and computed tomographic densities of liver, spleen, kidney and pancreas in beta-thalassaemia major.

Thirty-seven children with beta-thalassaemia major, eight children with liver cirrhosis, and 20 matched controls were enrolled in this study. Serum ferritin was determined in each subject by radio-immunoassay and liver enzymes by standard methods. The liver, spleen, kidney and pancreas densities were obtained by computed tomography using a Siemens Somatom 2 Scanner with 8-mm slice thickness. The iron content of liver biopsies from 10 patients was graded by staining. The mean serum ferritin of the thalassaemic patients was significantly higher than that of the control group (p = 0.0001). The ferritin of patients with cirrhosis and Wilson's disease was similar to that of the control group. The liver density of the thalassaemic patients was significantly higher than that of the control group (p less than 0.0001) while that of patients with liver cirrhosis and Wilson's disease was similar to the control group. The liver iron content of patients with liver cirrhosis was within the normal range. The spleen and kidney densities of patients with thalassaemia were higher than that of the control group with p values of 0.02 and 0.056, respectively. The density of the pancreas in patients with thalassaemia was not significantly different from that of the control group, (p = 0.52). There was correlation between the liver density and serum ferritin in patients with thalassaemia (r = 0.432, p less than 0.01) while there was no correlation between spleen, pancreas and kidney densities with serum ferritin.

Ultrasound findings in childhood chronic liver parenchymal diseases. An analysis of 41 patients.

Forty-one children with liver disease were studied by ultrasound scan at King Khalid University Hospital, Riyadh, Saudi Arabia. Diagnoses were confirmed either by liver biopsy or specific laboratory tests. Sonograms were studied for liver size, beam penetration, echogenicity, vascularity, and biliary tree abnormalities. Different liver diseases, such as chronic hepatitis, biliary cirrhosis, Wilson's disease, familial idiopathic cirrhosis, type III glycogen storage disease, and secondary haemochromatosis revealed non-specific disease patterns. Four cases of biliary cirrhosis and two cases of glycogen storage disease showed periportal fibrosis. Two cases of familial idiopathic cirrhosis and a case of Wilson's disease revealed thickening of the gall bladder wall, which has not been described in the literature.

Coagulation changes in sickle cell disease in early childhood.

Measurements of the coagulation system were carried out in children with sickle cell disease (SCD) in both steady state and on the 1st day of painful crisis and were compared to age- and sex-matched healthy controls. No significant differences were found in prothrombin time, partial thromboplastin time, thrombin time, reptilase time, plasma fibrinogen, antithrombin III, factor VIII:C, ristocetin-cofactor (Ri-Cof) and platelet aggregation responses to ADP, collagen and adrenaline. Abnormal aggregation responses to ristocetin were noted in all patients with SCD when compared to controls. Daily measurements during the first 4 days of painful crisis showed significant elevation of fibrinogen and Ri-Cof and enhancement of aggregation to ADP and adrenaline by the 3rd day of crisis. It was concluded that the changes noted, rather than being primarily responsible for the onset of crisis, can only be secondary changes arising from the aetiological factors of crisis, i.e. stasis and acute-phase proteins.

Prophylaxis of pneumococcal infection in sickle-cell disease by the combined use of vaccination and penicillin.

Forty children homozygotes for sickle-cell disease (SCD) aged 2-5 years were enrolled in a study to assess the efficacy of pneumococcal vaccine combined with penicillin prophylaxis in preventing pneumococcal infection. The vaccine was given initially and then every 2 years as a booster. In addition to pneumococcal vaccine, 24 children were prescribed penicillin-V orally twice daily while the remaining 16 were given benzathine penicillin intramuscularly every 4 weeks in the clinic. The study period lasted for 4 years during which the clinic attendance rate of both groups was 90% and 92% respectively. The compliance with oral penicillin intake was checked by random urine testing for penicillin. The test was positive in 128 out of 320 urine samples (40%). The parenteral penicillin was given at every clinic visit and the rate of compliance was 92%. Twelve episodes of bacterial sepsis were documented: 10 of them were caused by Gram-negative organisms. The pneumococcus caused a fatal episode of septicaemia and meningitis in a child who did not comply with penicillin prophylaxis. In a retrospective search, 15 episodes of bacterial sepsis have been documented in 22 children receiving no prophylaxis for 2 years. Eight of those episodes were caused by the pneumococcus. The combination of pneumococcal vaccine with penicillin prophylaxis was effective in preventing pneumococcal sepsis in children with SCD followed up for 153 patients years.