The Search for Cancer Biomarkers: Assessing the Distribution of INDEL Markers in Different Genetic Ancestries.

Cancer is a multifactorial group of diseases, being highly incident and one of the leading causes of death worldwide. In Brazil, there is a great variation in cancer incidence and impact among the different geographic regions, partly due to the genetic heterogeneity of the population in this country, composed mainly by European (EUR), Native American (NAM), African (AFR), and Asian (ASN) ancestries. Among different populations, genetic markers commonly present diverse allelic frequencies, but in admixed populations, such as the Brazilian population, data is still limited, which is an issue that might influence cancer incidence. Therefore, we analyzed the allelic and genotypic distribution of 12 INDEL polymorphisms of interest in populations from the five Brazilian geographic regions and in populations representing EUR, NAM, AFR, and ASN, as well as tissue expression in silico. Genotypes were obtained by multiplex PCR and the statistical analyses were done using R, while data of tissue expression for each marker was extracted from GTEx portal. We highlight that all analyzed markers presented statistical differences in at least one of the population comparisons, and that we found 39 tissues to be differentially expressed depending on the genotype. Here, we point out the differences in genotype distribution and gene expression of potential biomarkers for risk of cancer development and we reinforce the importance of this type of study in populations with different genetic backgrounds.

Association between forest resources and water availability: temporal analysis of the Serra Azul stream sub-basin.

Vegetation is expected to influence processes in the water cycle through its structural effects on key ecosystem functions in watersheds. However tropical forests are being submitted to anthropogenic pressures that result in great disturbances in the functioning of ecosystem services. Thus, the present study uses a landscape scale analysis for exploring the associations between land use changes and water availability in the Serra Azul stream watershed. The land use transitions from years 2013 to 2018 were investigated and a set of robust landscape metrics were analyzed across the study region, including water bodies Permanent Preservation Areas. A correlation analysis between the water volume of the Serra Azul reservoir and the landscape metrics was also performed to verify the association between forest resources and water availability. The results show that the region has been submitted to several impacts associated with the loss of forest areas resulting from landscape transformations throughout the region. Forest fragmentation associated to loss of connectivity severely limit water resources availability besides reducing the basin environmental resilience. The role of different management instruments for water resources protection was also discussed, emphasizing the need for participation of stakeholder in the creation process of these environmental protection instruments.

Estimating Asian Contribution to the Brazilian Population: A New Application of a Validated Set of 61 Ancestry Informative Markers.

Estimates of different ancestral proportions in admixed populations are very important in population genetics studies, especially for the detection of population substructure effects in studies of case-control associations. Brazil is one of the most heterogeneous countries in the world, both from a socio-cultural and a genetic point of view. In this work, we investigated a previously developed set of 61 ancestry informative markers (AIM), aiming to estimate the proportions of four different ancestral groups (African, European, Native American and Asian) in Brazilian populations. To the best of our knowledge, this is the first study to use a set of AIM to investigate the genetic contribution of all four main parental populations to the Brazilian population, including Asian contribution. All selected markers were genotyped through multiplex PCR and capillary electrophoresis. The set was able to successfully differentiate the four ancestral populations (represented by 939 individuals) and identify their genetic contributions to the Brazilian population. In addition, it was used to estimate individual interethnic admixture of 1050 individuals from the Southeast region of Brazil and it showed that these individuals present a higher European ancestry contribution, followed by African, Asian and Native American ancestry contributions. Therefore, the 61 AIM set has proved to be a valuable tool to estimate individual and global ancestry proportions in populations mainly formed by these four groups. Our findings highlight the importance of using sets of AIM to evaluate population substructure in studies carried in admixed populations, in order to avoid misinterpretation of results.

Addition of cattle manure to sheep bedding allows vermicomposting process and improves vermicompost quality.

Animal waste is usually a good substrate for vermicomposting. However, numerous animal husbandry systems use bedding that consists primarily of lignocellulosic substrates, which hinders earthworm and microorganism's development and thus, the entire bioconversion process. One possible solution is to mix the used bedding with other waste materials that are more amenable to earthworm ingestion and can provide better conditions for earthworm population growth. Here, we have aimed to examine the effectiveness of such procedure by mixing rice-husk-based sheep bedding with cattle manure in different proportions (0%, 25%, 50%, 75% and 100%). We have carried out vermicomposting experiments in benchtop vermireactors inoculated with 0.88kg of dry matter (sheep bedding+cattle manure). Data used in the Principal Component Analysis were the multiple vermicomposting variables (i.e., EC; pH; HA/FA and C/N ratios; P, K, cellulose, and hemicellulose content). The effect of the treatment on earthworm count was analyzed with ANOVA. We have observed that the addition of at least 25% of cattle manure to sheep bedding allows vermicomposting process but it is necessary 148days to obtain a stabilized vermicompost. However, increasing the proportion of cattle manure to sheep bedding, the vermicomposting time decreases proportionally to 94days. We concluded that vermicomposting can be considered a bioprocess to stabilize rice husk after being used as sheep bedding.

Distribution of allelic and genotypic frequencies of IL1A, IL4, NFKB1 and PAR1 variants in Native American, African, European and Brazilian populations.

BACKGROUND: The inflammatory response plays a key role at different stages of cancer development. Allelic variants of the interleukin 1A (IL1A), interleukin 4 (IL4), nuclear factor kappa B1 (NFKB1) and protease-activated receptor 1 (PAR1) genes may influence not only the inflammatory response but also susceptibility to cancer development. Among major ethnic or continental groups, these polymorphic variants present different allelic frequencies. In admixed populations, such as the Brazilian population, data on distribution of these polymorphisms are limited. Here, we collected samples of cancer-free individuals from the north, northeast, midwest, south and southeast regions of Brazil and from the three main groups that gave rise to the Brazilian population: Native Americans from the Brazilian Amazon, Africans and Europeans. We describe the allelic distributions of four IL1A (rs3783553), IL4 (rs79071878), NFKB1 (rs28362491) and PAR1 (rs11267092) gene polymorphisms, which the literature describes as polymorphisms with a risk of cancer or worse prognosis for cancer. RESULTS: The genotypic distribution of the four polymorphisms was statistically distinct between Native Americans, Africans and Europeans. For the allelic frequency of these polymorphisms, the Native American population was the most distinct among the three parental populations, and it included the greatest number of alleles with a risk of cancer or worse prognosis for cancer. The PAR1 gene polymorphism allelic distribution was similar among all Brazilian regions. For the other three markers, the northern region population was statistically distinct from other Brazilian region populations. CONCLUSION: The IL1A, IL4, NFKB1 and PAR1 gene polymorphism allelic distributions are homogeneous among the regional Brazilian populations, except for the northern region, which significantly differs from the other four Brazilian regions. Among the parental populations, the Native American population exhibited a higher incidence of alleles with risk of cancer or worse prognosis for cancer, which can indicate greater susceptibility to this disease. These genetic data may be useful for future studies on the association between these polymorphisms and cancer in the investigated populations.

Phylogenetic analysis of canine distemper virus in South America clade 1 reveals unique molecular signatures of the local epidemic.

Canine distemper virus (CDV) is a highly contagious pathogen for domestic dogs and several wild carnivore species. In Brazil, natural infection of CDV in dogs is very high due to the large non-vaccinated dog population, a scenario that calls for new studies on the molecular epidemiology. This study investigates the phylodynamics and amino-acid signatures of CDV epidemic in South America by analyzing a large dataset compiled from publicly available sequences and also by collecting new samples from Brazil. A population of 175 dogs with canine distemper (CD) signs was sampled, from which 89 were positive for CDV, generating 42 new CDV sequences. Phylogenetic analysis of the new and publicly available sequences revealed that Brazilian sequences mainly clustered in South America 1 (SA1) clade, which has its origin estimated to the late 1980's. The reconstruction of the demographic history in SA1 clade showed an epidemic expanding until the recent years, doubling in size every nine years. SA1 clade epidemic distinguished from the world CDV epidemic by the emergence of the R580Q strain, a very rare and potentially detrimental substitution in the viral genome. The R580Q substitution was estimated to have happened in one single evolutionary step in the epidemic history in SA1 clade, emerging shortly after introduction to the continent. Moreover, a high prevalence (11.9%) of the Y549H mutation was observed among the domestic dogs sampled here. This finding was associated (p<0.05) with outcome-death and higher frequency in mixed-breed dogs, the later being an indicator of a continuous exchange of CDV strains circulating among wild carnivores and domestic dogs. The results reported here highlight the diversity of the worldwide CDV epidemic and reveal local features that can be valuable for combating the disease.

Amerindian genetic ancestry and INDEL polymorphisms associated with susceptibility of childhood B-cell Leukemia in an admixed population from the Brazilian Amazon.

Acute lymphoblastic leukemia (ALL) is a malignant tumor common in children. Studies of genetic susceptibility to cancer using biallelic insertion/deletion (INDEL) type polymorphisms associated with cancer development pathways may help to clarify etymology of ALL. In this study, we investigate the role of eight functional INDEL polymorphisms and influence of genetic ancestry to B-cell ALL susceptibility in children of Brazilian Amazon population, which has a high degree of inter-ethnic admixture. Ancestry analysis was estimated using a panel of 48 autosomal ancestry informative markers. 130 B-cell ALL patients and 125 healthy controls were included in this study. The odds ratios and 95% confidence intervals were adjusted for confounders. The results indicated an association between the investigated INDEL polymorphisms in CASP8 (rs3834129), CYP19A1 (rs11575899) e XRCC1 (rs3213239) genes in the development of B-cell ALL. The carriers of Insertion/Insertion (Ins/Ins) genotype of the polymorphism in CASP8 gene presented reduced chances of developing B-cell ALL (P=0.001; OR=0.353; 95% CI=0.192-0.651). The Deletion/Deletion (Del/Del) genotype of the polymorphism in CYP19A1 gene was associated to a lower chance of developing B-cell ALL (P=3.35×10-6; OR=0.121; 95% CI=0.050-0.295), while Del/Del genotype of the polymorphism in XRCC1 gene was associated to a higher chance of developing B-cell ALL (P=2.01×10-4; OR=6.559; 95% CI=2.433-17.681). We also found that Amerindian ancestry correlates with the risk of B-cell ALL. For each increase of 10% in the Amerindian ancestry results in 1.4-fold chances of developing B-cell ALL (OR=1.406; 95% IC=1.123-1.761), while each increase of 10% in the European ancestry presents a protection effect in the development of B-cell ALL (OR=0.666; 95% IC=0.536-0.827). The results suggest that genetic factors influence leukemogenesis and might be explored in the stratification of B-cell ALL risk in admixed populations.

The galE gene encoding the UDP-galactose 4-epimerase of Brevibacterium lactofermentum is coupled transcriptionally to the dmdR gene.

The galE gene of Brevibacterium lactofermentum, encoding UDP-galactose 4-epimerase (EC 5.1.3.2), has been identified by DNA sequencing downstream from the orf1-sigB-dmdR region. The arrangement of the sigB-dtxR-galE cluster is also conserved in Corynebacterium diphtheriae. The deduced galE product was a protein of 329 aa residues (35.4 kDa) that shared a high degree of identity to known UDP-galactose 4-epimerase proteins from Gram-positive microorganisms (Streptomyces lividans and Streptococcus thermophilus). Transcriptional analysis of the dmdR and galE genes in nutrient-rich medium showed that these genes are part of an operon, that is actively transcribed as a bicistronic mRNA during the exponential growth phase, but transcription of the operon is decreased during the stationary growth phase. In addition, the dmdR gene was also expressed as a monocistronic 0.7-kb transcript during the active growth phase.

Three genes hrdB, hrdD and hrdT of Streptomyces griseus IMRU 3570, encoding sigma factor-like proteins, are differentially expressed under specific nutritional conditions.

Three genes (hrd) homologous to the rpoD gene of Escherichia coli, that encode sigma factor-like proteins, have been cloned from DNA of the candicidin-producing strain Streptomyces griseus IMRU 3570. They are located in different regions of the chromosome. Sequence analysis showed that the first one is analogous to the hrdB gene of S. coelicolor. The second showed high similarity to the hrdD gene of S. coelicolor and S. aureofaciens and is linked, as in S. coelicolor, to a N-acetyltransferase-encoding gene (nat) distantly related to the pat and bar genes that encode resistance to bialafos. The third showed no close homology with other known hrd genes from actinomycetes and has been named hrdT. Functional domains in the three S. griseus Hrd proteins are highly conserved in relation to those of the sigma 70 protein family. Northern analysis showed that hrdB is expressed as a 1.9-kb transcript during active growth in phosphate-rich medium, but it is less efficiently transcribed under sporulation conditions (phosphate-starved) or after a heat-shock treatment. Two other shorter transcripts of 1.2 and 0.7 kb were also detected with the same probe. The hrdD gene is transcribed as a single 1.1-kb transcript under sporulation conditions following nutritional shiftdown and, to a lower extent, during growth conditions in phosphate-rich medium. The hrdT gene is weakly transcribed (1.5-kb RNA) under all conditions tested. The hrd-encoded sigma factors probably recognize actinomycetes promoters (SEP type) with E. coli-like consensus sequences.

Transcriptional analysis of the sigA and sigB genes of Brevibacterium lactofermentum.

Transcription of the sigA and sigB genes of Brevibacterium lactofermentum, encoding the principal sigma factors of RNA polymerase, has been investigated by Northern blot and primer extension analysis. Northern hybridizations revealed that sigA is transcribed as a monocistronic mRNA of 1.7 kb and sigB gives two transcripts of 1.1 and 1.5 kb. Similar transcription patterns of sigA and sigB genes in nutrient-rich medium were observed; transcripts of both genes occurred simultaneously throughout the exponential growth phase and decayed clearly when the stationary phase was reached. Primer extension analysis of B. lactofermentum RNA showed that the sigA and sigB transcription initiation sites are located 17 bp and 24 bp upstream from the first nucleotide of the respective translation initiation codons. Alignment of the sigA and sigB promoter regions provided evidence for highly conserved sequences in both of them.

A moderate amplification of the mecB gene encoding cystathionine-gamma-lyase stimulates cephalosporin biosynthesis in Acremonium chrysogenum.

L-cysteine is a precursor of the penicillin, cephalosporin and cephamycin families of beta-lactam antibiotics. Cystathionine-gamma-lyase (encoded by the mecB gene), an enzyme that splits cystathionine releasing cysteine, is required for high-level cephalosporin production in methionine-supplemented medium. By amplification of the mecB gene in Acremonium chrysogenum C10, several transformants were obtained that produced 10-40% higher levels of cephalosporin. All selected transformants contained at least two or three copies of the mecB gene as shown by Southern hybridization with a probe internal to mecB. Two of these transformants, A. chrysogenum T27 and A. chrysogenum T58, showed 4- to 10-fold higher cystathionine-gamma-lyase activity than the control strain. Northern hybridizations indicated that the levels of the two mecB transcripts of 1.7 and 1.5 kb were greatly increased in transformants T27 and T58. Fermentor studies using controlled conditions confirmed that transformant T27 was a cephalosporin overproducer, reaching titers of nearly 2000 microg/ml of cephalosporin in Shen-defined medium that correlated with two- to fourfold higher cystathionine-gamma-lyase levels than in the control strain. Transformant T58 containing five- to sixfold higher levels of cystathionine-gamma-lyase in fermentor cultures showed a reduced growth rate and a slow cephalosporin accumulation rate. In conclusion, moderately increased levels of cystathionine-gamma-lyase stimulated cephalosporin production but very high levels of this enzyme were deleterious for growth and cephalosporin biosynthesis.

Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae dtxR homolog from Brevibacterium lactofermentum.

A homolog of the Corynebacterium diphtheriae dtxR gene was isolated from Brevibacterium lactofermentum. The product of the B. lactofermentum dtxR gene was immunoreactive with polyclonal anti-DtxR antibodies and functioned as an iron-activated repressor capable of regulating the expression of beta-galactosidase from a diphtheria tox promoter/operator transcriptional fusion in recombinant Escherichia coli. The extents of induction by increasing concentrations of the chelator 2,2'-dipyridyl were identical in cells expressing DtxR from either C. diphtheriae or B. lactofermentum.

Multiple sigma factor genes in Brevibacterium lactofermentum: characterization of sigA and sigB.

Four rpoD hybridizing signals have been identified in the chromosome of Brevibacterium lactofermentum. Two rpoD-like genes, sigA and sigB, have been cloned and sequenced, and they encode principal sigma factors of the RNA polymerase. The deduced amino acid sequences of SigA and SigB showed very high similarities to those of Mycobacterium smegmatis MysA and MysB proteins, respectively, and also to those of HrdB proteins from different Streptomyces species. SigA and SigB maintain the conserved motifs of sigma 70-like principal sigma factors. sigB is closely linked to the dtxR gene (encoding a repressor of iron-regulated promoters homologous to the diphtheria toxin repressor from Corynebacterium diphtheriae.

Cloning and characterization of an alpha-amylase gene from Streptomyces sp WL6.

An alpha-amylase gene from Streptomyces sp WL6 was cloned on a 3.1kb DNA fragment, which was completely sequenced. The 3088 nucleotide sequence obtained contains three putative coding regions in the same orientation. The one corresponding to the structural region of the alpha-amylase gene has a deduced amino acid sequence of 459 residues, showing up to 71% identity to other alpha-amylases. An incomplete ORF was identified upstream the alpha-amylase gene, and the deduced product presents some homology to proteins involved in catabolic regulation.

Penicillin and cephalosporin biosynthesis: mechanism of carbon catabolite regulation of penicillin production.

Penicillins and cephalosporins are synthesized by a series of enzymatic reactions that form the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and convert this tripeptide into the final penicillin or cephalosporin molecules. One of the enzymes, isopenicillin N synthase has been crystallyzed and its active center identified. The three genes pcbAB, pcbC and penDE involved in penicillin biosynthesis are clustered in Penicillium chrysogenum, Aspergillus nidulans and Penicillium nalgiovense. Carbon catabolite regulation of penicillin biosynthesis is exerted by glucose and other easily utilizable carbon sources but not by lactose. The glucose effect is enhanced by high phosphate concentrations. Glucose represses the biosynthesis of penicillin by preventing the formation of the penicillin biosynthesis enzymes. Transcription of the pcbAB, pcbC and penDE genes of P. chrysogenum is strongly repressed by glucose and the repression is not reversed by alkaline pHs. Carbon catabolite repression of penicillin biosynthesis in A. nidulans is not mediated by CreA and the same appears to be true in P. chrysogenum. The first two genes of the penicillin pathway (pcbAB and pcbC) are expressed from a bidirectional promoter region. Analysis of different DNA fragments of this bidirectional promoter region revealed two important DNA sequences (boxes A and B) for expression and glucose catabolite regulation of the pcbAB gene. Using protein extracts from mycelia grown under carbon catabolite repressing or derepressing conditions DNA-binding proteins that interact with the bidirectional promoter region were purified to near homogeneity.

Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum.

Five mutants of Penicillium chrysogenum blocked in penicillin biosynthesis (npe) which are deficient in isopenicillin N-acyltransferase were isolated previously. Three of these mutants, npe6, npe7, and npe8, have been characterized at the molecular level and compared with npe10, a deletion mutant. Transcripts of normal size (1.15 kb) of the penDE genes, which encode isopenicillin N-acyltransferase, and also of the pcbAB (11.5 kb) and pcbC (1.1 kb) genes were observed in all mutants except for the npe10 mutant. Immunoblotting studies using antibodies against isopenicillin N-acyltransferase showed that all mutants (except npe10) formed the 40-kDa (unprocessed) protein and the 29-kDa subunit of the isopenicillin N-acyltransferase. The 11-kDa subunit could not be observed in the immunoblots. The mutant penDE genes of strains npe6, npe7, and npe8 were cloned and sequenced. These three strains showed a mutation in the penDE genes which results in a single amino acid change in each modified isopenicillin N-acyltransferase. The mutation in npe6 resulted in a change of Gly-150 to Val, whereas the mutation in both npe7 and npe8 introduced a change of Glu-258 to Lys. Replacement of the Val-150 and Lys-258 mutations by constructing hybrid isopenicillin N-acyltransferase molecules led to the recovery of the isopenicillin N-acyltransferase activity. The mutations in npe6, npe7, and npe8 do not affect the ability of the 40-kDa isopenicillin N-acyltransferase to be processed into the component subunits.

Expression of genes and processing of enzymes for the biosynthesis of penicillins and cephalosporins.

The genes pcbAB, pcbC and penDE encoding the enzymes (alpha-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase and isopenicillin N acyltransferase, respectively) involved in the biosynthesis of penicillin have been cloned from Penicillin chrysogenum and Aspergillus nidulans. They are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of Penicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. Each gene is expressed as a single transcript from separate promoters. Enzyme regulation studies and gene expression analysis have provided useful information to understand the control of genes involved in penicillin biosynthesis. The enzyme isopenicillin N acyltransferase encoded by the penDE gene is synthesized as a 40 kDa protein that is (self)processed into two subunits of 29 and 11 kDa. Both subunits appear to be required for acyl-CoA 6-APA acyltransferase activity. The isopenicillin N acyltransferase was shown to be located in microbodies, whereas the isopenicillin N synthase has been reported to be present in vesicles of the Golgi body and in the cell wall. A mutant in the carboxyl-terminal region of the isopenicillin N acyltransferase lacking the three final amino acids of the enzymes was not properly located in the microbodies and failed to synthesize penicillin in vivo. In C. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes (alpha-aminoadipyl-cysteinyl) valine synthetase and isopenicillin N synthase) of the cephalosporin pathway which are very similar to those involved in penicillin biosynthesis. Cluster II (cefEF-cefG), encodes the last three enzymatic activities (deacetoxycephalosporin C synthetase/hydroxylase and deacetylcephalosporin C acetyltransferase) of the cephalosporin pathway. It is unknown, at this time, if the cefD gene encoding isopenicillin epimerase is linked to any of these two clusters. Methionine stimulates cephalosporin biosynthesis in cultures of three different strains of A. chrysogenum. Methionine increases the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC and cefG respectively) which are separated in the two different clusters of cephalosporin biosynthesis genes. This result suggests that both clusters of genes have regulatory elements which are activated by methionine. Methionine-supplemented cells showed higher levels of transcripts of the pcbAB, pcbC, cefEF genes and to a lesser extent of cefG than cells grown in absence of methionine. The levels of the cefG transcript were very low as compared to those of pcbAB, pcbC and cefEF.(ABSTRACT TRUNCATED AT 400 WORDS)

Exogenous methionine increases levels of mRNAs transcribed from pcbAB, pcbC, and cefEF genes, encoding enzymes of the cephalosporin biosynthetic pathway, in Acremonium chrysogenum.

Methionine stimulated cephalosporin production in cultures of three different strains of Acremonium chrysogenum when added either at inoculation time or at 72 h to cells grown previously in the absence of methionine. When methionine was added at 72 h, the stimulation of cephalosporin biosynthesis was observed only 12 h later and required de novo protein synthesis. Methionine increased the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC and cefG, respectively) located in the two clusters of cephalosporin biosynthesis genes in the wild-type A. chrysogenum strain and also in the two improved strains, CW19 and C10. Methionine-supplemented cells showed higher levels of transcripts of the four known genes (pcbAB, pcbC, cefEF and, to a slight extent, cefG) of the cephalosporin biosynthetic pathway than cells grown in the absence of methionine. The levels of the cefG transcript were much lower than those of the pcbAB, pcbC, and cefEF transcripts. The induction by methionine of transcription of the four cephalosporin biosynthesis genes and the known effect of this amino acid on the differentiation of A. chrysogenum indicate that methionine exerts a pleiotropic effect that coordinately regulates cephalosporin biosynthesis and differentiation.

Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-gamma-lyase.

In Acremonium chrysogenum, biosynthesis of cysteine for the formation of cephalosporin has been proposed to occur through the reverse transsulfuration pathway. A gene, named mecB, has been cloned from an A. chrysogenum C10 genomic library in lambdaEMBL3-ble. The cloned DNA fragment encodes a protein of 423 amino acids with a deduced molecular mass of 45 kDa that shows great similarity to cystathionine-gamma-lyases from Saccharomyces cerevisiae and other eukaryotic organisms. The protein was shown to be functional because it restores growth on methionine to A. nidulans C47 (mecB10), a mutant that is known to be defective in cystathionine-gamma-lyase. The cloned gene did not complement A. nidulans mecA or metG mutants. Enzyme activity assays confirmed that the cloned mecB gene encodes a cystathionine-gamma-lyase activity. The mecB gene is present in a single copy in the wild-type A. chrysogenum (Brotzu's strain) and also in the A. chrysogenum strain C10, a high cephalosporin producer. The gene is localized on chromosome VIII (5.3 Mb), as shown by hybridization to A. chrysogenum chromosomes resolved by pulsed-field gel electrophoresis. Transcription of the mecB gene gives rise to a major transcript of 1.5 kb and a minor one of 1.7 kb. The transcript levels were not significantly affected by addition of DL-methionine to the culture, indicating that expression of this gene is not regulated by methionine. The availability of this gene provides a very useful tool for understanding the proposed role of cystathionine-gamma-lyase in splitting cystathionine to supply cysteine for cephalosporin biosynthesis.

The gene encoding gamma-actin from the cephalosporin producer Acremonium chrysogenum.

The nucleotide sequence of a 3240-bp genomic fragment including the gamma-actin-encoding gene from Acremonium chrysogenum has been determined, showing an open reading frame of 1691 bp, interrupted by five introns with fungal consensus splice-site junctions. The untranslated regions of the actA gene contain a consensus TATA box, a CCAAT motif, pyrimidine stretches and the polyadenylation sequence AATAA. The predicted protein (375 amino acids) revealed high identity to gamma-actins from fungi (> 90%). Gene phylogenies constructed using DNA and protein sequences support the grouping of A. chrysogenum actin close to those from the majority of the filamentous fungi. The actA gene is present as a single copy in the genome of A. chrysogenum; and its expression level, opposite to pcbC and cefEF cephalosporin biosynthetic genes, was steady during cephalosporin fermentation, showing a single 1.4-kb transcript.