Strain AP1 Research Articles

Assessing the therapeutic potential of long-chain isomaltooligosaccharides in diabetic and hyperlipidemic rats

BackgroundThe global rise in diabetes prevalence necessitates effective treatments. Rats, mimicking physiological changes seen in Type 2 diabetes, serve as valuable models for studying metabolic disorders. Natural health supplements, especially prebiotics, are gaining interest for improving metabolic health. Isomaltooligosaccharides (IMOs), classified as functional oligosaccharides and prebiotics, have attracted attention due to their beneficial effects on gut microbiota balance and cholesterol reduction. However, commercial IMOs often contain undesirable sugars, leading to the development of long-chain IMOs with enhanced prebiotic properties.MethodsThis study assessed the therapeutic potential of long-chain IMOs derived from Bacillus subtilis strain AP-1 compared to inulin, a widely recognized prebiotic, in addressing hyperglycemia and hyperlipidemia in rats.ResultsIMOs treatment effectively reduced blood sugar and triglyceride levels similarly to inulin supplementation. Proteomic analysis revealed changes in hepatic protein profiles, with upregulated pathways including glutathione metabolism, oxidative phosphorylation, and pentose and glucuronate interconversion, while pathways related to fatty acid and amino acid biosynthesis exhibited downregulation. These results suggest promising therapeutic effects of IMOs treatment on diabetes and hyperlipidemia by influencing key metabolic pathways.ConclusionsOur findings highlight the potential of long-chain IMOs as targeted interventions for metabolic disorders, warranting further investigation into their clinical applicability and mechanisms of action.

Aureobasidium pullulans formulations: evaluation of the effectiveness against grey mould of table grape

Antagonism against Botrytis cinerea is often carried out using yeast as direct antagonists. Aureobasidium pullulans strain AP1 was tested in two different formulations: wettable powder (WP) and oil dispersion (OD). By in vitro assays, the viability of the strain cells was constantly evaluated for seven months and the OD formulation ensured the highest cells viability. The efficacy of the formulations was assayed by evaluating the production of volatile and non-volatile metabolites. Results showed that the formulation affected the non-volatile less than the volatile metabolites. Both AP1 WP and AP1 OD non-volatile metabolites displayed almost 50% of mycelial pathogen inhibition. Comparing the two products, the lowest EC50 value (518.15 mg L− 1) was detected for the AP1 OD formulation that was thus chosen for postharvest in vivo assays. The preventative treatments (200, 400, 800 mg L− 1) were active in reducing the pathogen incidence on table grape on average by 52%. Instead, in the curative application assay, the highest concentration (800 mg L− 1) reduced grey mold incidence by 86%. The present study reported the potential of two new formulations to use against the postharvest grey mold of table grape for a possible further commercial product development.

Characterization of a Filamentous Phage, Vaf1, from Vibrio alginolyticus AP-1.

Filamentous phages are ubiquitously distributed in the global oceans. However, little is known about their biological contribution to their host's genetic and phenotypic diversity. In this study, a filamentous phage, Vaf1, was isolated and characterized from the emerging marine pathogen strain Vibrio alginolyticus AP-1. We explored the effects of the resident phage Vaf1 on the host physiology under diverse conditions by precisely deleting the entire phage Vaf1. Our results demonstrate that the presence of phage Vaf1 significantly increased biofilm formation, swarming motility, and contact-dependent competition. Furthermore, the gene expression profile suggests that several phage genes were upregulated in response to low-nutrient conditions. Unexpectedly, an in vivo study of zebrafish shows that fish infected with strain ΔVaf1 survived longer than those infected with wild-type strain AP-1, indicating that Vaf1 contributes to the virulence of V. alginolyticus. Together, our results provide direct evidence for the effect of Vaf1 phage-mediated phenotypic changes in marine bacteria V. alginolyticus. This further emphasizes the impressive complexity and diversity that filamentous phage-host interactions pose and the challenges associated with bacterial disease control in marine aquaculture. IMPORTANCE Non-lytic filamentous phages can replicate without killing their host, establishing long-term persistence within the bacterial host. In contrast to the well-studied CTXφ phage of the human-pathogenic Vibrio cholerae, little is known about the filamentous phage Vaf1 and its biological role in host fitness. In this study, we constructed a filamentous phage-deleted strain, ΔVaf1, and provided direct evidence on how an intact phage, φVaf1, belonging to the family Inoviridae, helps the bacterial host AP-1 to overcome adverse environmental conditions. Our results likely open new avenues for fundamental studies on how filamentous phage-host interactions regulate different aspects of Vibrio cell behaviors.

Production of a Series of Long-Chain Isomaltooligosaccharides from Maltose by Bacillus subtilis AP-1 and Associated Prebiotic Properties.

Bacillus subtilis strain AP-1, which produces α-glucosidase with transglucosidase activity, was used to produce a series of long-chain isomaltooligosaccharides (IMOs) with degree of polymerization (DP) ranging from 2 to 14 by direct fermentation of maltose. A total IMOs yield of 36.33 g/L without contabacillusmination from glucose and maltose was achieved at 36 h of cultivation using 50 g/L of maltose, with a yield of 72.7%. IMOs were purified by size exclusion chromatography with a Superdex 30 Increase column. The molecular mass and DP of IMOs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Subsequently, linkages in produced oligosaccharides were verified by enzymatic hydrolysis with α-amylase and oligo-α-1,6-glucosidase. These IMOs showed prebiotic properties, namely tolerance to acidic conditions and digestive enzymes of the gastrointestinal tract, stimulation of probiotic bacteria growth to produce short-chain fatty acids and no stimulating effect on pathogenic bacteria growth. Moreover, these IMOs were not toxic to mammalian cells at up to 5 mg/mL, indicating their biocompatibility. Therefore, this research demonstrated a simple and economical method for producing IMOs with DP2-14 without additional operations; moreover, the excellent prebiotic properties of the IMOs offer great prospects for their application in functional foods.

Two Novel Genomospecies in the Agrobacterium tumefaciens Species Complex Associated with Rose Crown Gall.

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD, gyrB, recA, and rpoB), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as "A. viscosum" (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named "G19" and "G20." Hence, we propose the strains Rew, Rnw, and Rnr as the members of "G19" and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

Early Lymphocyte Loss and Increased Granulocyte/Lymphocyte Ratio Predict Systemic Spread of Streptococcus pyogenes in a Mouse Model of Acute Skin Infection.

Background: Group A streptococci may induce lymphopenia, but the value of lymphocyte loss as early biomarkers for systemic spread and severe infection has not been examined systematically.Methods: We evaluated peripheral blood cell indices as biomarkers for severity and spread of infection in a mouse model of Streptococcus pyogenes skin infection, using two isolates of greatly differing virulence. Internal organs were examined histologically.Results: After subcutaneous inoculation, strain AP1 disseminated rapidly to peripheral blood and internal organs, causing frank sepsis. In contrast, seeding of internal organs by 5448 was mild, this strain could not be isolated from blood, and infection remained mostly localized to skin. Histopathologic examination of liver revealed microvesicular fatty change (steatosis) in AP1 infection, and examination of spleen showed elevated apoptosis and blurring of the white pulp/red pulp border late (40 h post infection) in AP1 infection. Both strains caused profound lymphopenia, but lymphocyte loss was more rapid early in AP1 infection, and lymphocyte count at 6 h post infection was the most accurate early marker for AP1 infection (area under the receiver operator curve [AUC] = 0.93), followed by the granulocyte/lymphocyte ratio (AUC = 0.89).Conclusions: The results suggest that virulence of S. pyogenes correlates with the degree of early lymphopenia and underscore the value of peripheral blood indices to predict severity of bacterial infections in mice. Early lymphopenia and elevated granulocyte/lymphocyte ratio merit further investigation as biomarkers for systemic spread of S. pyogenes skin infections in humans and, possibly, related pyogenic streptococci in humans and animals.

Comparative Genomics of Streptococcus pyogenes M1 isolates differing in virulence and propensity to cause systemic infection in mice

Streptococcus pyogenes serotype M1 is a frequent cause of severe infections in humans. Some M1 isolates are pathogenic in mice and used in studies on infection pathogenesis. We observed marked differences in murine infections caused by M1 strain SF370, 5448, 5448AP or AP1 which prompted us to sequence the whole genome of isolates 5448 and AP1 for comparative analysis. Strain 5448 is known to acquire inactivating mutations in the CovRS two-component system during mouse infection, producing hypervirulent progeny such as 5448AP. Isolates AP1 and 5448AP, more than 5448, caused disseminating infections that became systemic and lethal. SF370 was not pathogenic. Phages caused gross genetic differences and increased the gene content of AP1 by 8% as compared to 5448 and SF370. Each of six examined M1 genomes contained two CRISPR-Cas systems. Phage insertion destroyed a type II CRISPR-Cas system in AP1 and other strains of serotypes M1, M3, M6 and M24, but not in M1 strains 5448, SF370, MGAS5005, A20 or M1 476. A resulting impaired defence against invading genetic elements could have led to the wealth of phages in AP1. AP1 lacks genetic features of the MGAS5005-like clonal complex including the streptodornase that drives selection for hypervirulent clones with inactivated CovRS system. Still, inactivating mutations in covS were a common genetic feature of AP1 and the MGAS5005-like isolate 5448AP. Abolished expression of the cysteine proteinase SpeB, due to CovRS inactivation could be a common cause for hypervirulence of the two isolates. Moreover, an additional protein H-coding gene and a mutation in the regulator gene rofA distinguished AP1 form other M1 isolates. In conclusion, hypervirulence of S. pyogenes M1 in mice is not limited to the MGAS5005-like genotype.

Genome sequence and description of Bacteroides timonensis sp. nov.

Bacteroides timonensis strain AP1T (= CSUR P194 = DSM 26083) is the type strain of B. timonensis sp. nov. This strain, whose genome is described here, was isolated from the fecal flora of a 21-year-old French Caucasoid female who suffered from severe anorexia nervosa. Bacteroides timonensis is a Gram-negative, obligate anaerobic bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 7,130,768 bp long genome (1 chromosome, no plasmid) exhibits a G+C content of 43.3% and contains 5,786 protein-coding and 59 RNA genes, including 2 rRNA genes.

Natronoflexus pectinivorans gen. nov. sp. nov., an obligately anaerobic and alkaliphilic fermentative member of Bacteroidetes from soda lakes

Anaerobic enrichment with pectin at pH 10 and moderate salinity inoculated with sediments from soda lakes of the Kulunda Steppe (Altai, Russia) resulted in the isolation of a novel member of the Bacteroidetes, strain AP1T. The cells are long, flexible, Gram-negative rods forming pink carotenoids. The isolate is an obligate anaerobe, fermenting various carbohydrates to acetate and succinate. It can hydrolyze and utilize pectin, xylan, starch, laminarin and pullulan as growth substrates. Growth is possible in a pH range from 8 to 10.5, with an optimum at pH 9.5, and at a salinity range from 0.1 to 2 M Na+. Phylogenetic analysis based on 16S rRNA sequences placed the isolate into the phylum Bacteroidetes as a separate lineage within the family Marinilabilaceae. On the basis of distinct phenotype and phylogeny, the soda lake isolate AP1T is proposed to be assigned in a new genus and species Natronoflexus pectinivorans (=DSM24179T = UNIQEM U807T).Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-011-0399-7) contains supplementary material, which is available to authorized users.

Actions of Mycobacterium sp. Strain AP1 on the Saturated- and Aromatic-Hydrocarbon Fractions of Fuel Oil in a Marine Medium

The pyrene-degrading Mycobacterium sp. strain AP1 grew in nutrient-supplemented artificial seawater with a heavy fuel oil as the sole carbon source, causing the complete removal of all linear (C(12) to C(40)) and branched alkanes from the aliphatic fraction, as well as an extensive degradation of the three- and four-ring polycyclic aromatic hydrocarbons (PAHs) phenanthrene (95%), anthracene (80%), fluoranthene (80%), pyrene (75%), and benzo(a)anthracene (30%). Alkylated PAHs, which are more abundant in crude oils than the nonsubstituted compounds, were selectively attacked at extents that varied from more than 90% for dimethylnaphthalenes, methylphenanthrenes, methylfluorenes, and methyldibenzothiophenes to about 30% for monomethylated fluoranthenes/pyrenes and trimethylated phenanthrenes and dibenzothiophenes. Identification of key metabolites indicated the utilization of phenanthrene, pyrene, and fluoranthene by known assimilatory metabolic routes, while other components were cooxidized. Detection of mono- and dimethylated phthalic acids demonstrated ring cleavage and further oxidation of alkyl PAHs. The extensive degradation of the alkanes, the two-, three-, and four-ring PAHs, and their 1-, 2-, and 3-methyl derivatives from a complex mixture of hydrocarbons by Mycobacterium sp. strain AP1 illustrates the great substrate versatility of alkane- and PAH-degrading mycobacteria.

Streptomyces marokkonensis sp. nov., isolated from rhizosphere soil of Argania spinosa L.

The novel actinomycete strain Ap1(T) was isolated from rhizosphere soil of the argan tree (Argania spinosa L.) in the south of Morocco. Strain Ap1(T) has been reported as a novel producer of the pentaene polyene macrolide isochainin, which strongly inhibits the growth of pathogenic yeasts and phytopathogenic fungi. Strain Ap1(T) shows a greyish-white aerial mycelium with chains of smooth-surfaced spores of the Spiralis type and a cell wall containing ll-diaminopimelic acid. Based on chemotaxonomy and morphological features, strain Ap1(T) was identified as a member of the genus Streptomyces. 16S rRNA gene sequence similarities based on almost-complete 16S rRNA gene sequences showed that strain Ap1(T) is closely associated with members of the Streptomyces violaceoruber species group (S. violaceoruber, S. coelescens, S. violaceorubidus, 'S. caesius', 'S. lividans', S. violaceolatus and S. humiferus) and others (Streptomyces aurantiogriseus, S. lienomycini, S. chattanoogensis, S. rubrogriseus and S. tendae). However, protein profiling, DNA-DNA hybridization and BOX-PCR fingerprinting proved a relationship above the species level. In addition, the phenotype also allowed for the differentiation of strain Ap1(T) from its closest neighbours. As a result of this polyphasic approach, we conclude that strain Ap1(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces marokkonensis sp. nov. is proposed. The type strain is Ap1(T) (=R-22003(T) =LMG 23016(T) =DSM 41918(T)).

Metabolism of fluoranthene by Mycobacterium sp. strain AP1

The pyrene-degrading Mycobacterium strain AP1 was found to utilize fluoranthene as a sole source of carbon and energy. Identification of metabolites formed from fluoranthene (by growing cells and washed-cell suspensions), the kinetics of metabolite accumulation, and metabolite-feeding studies all indicated that strain AP1 oxidizes fluoranthene using three alternative routes. The first route is initiated by dioxygenation at C-7 and C-8 and, following meta cleavage and pyruvate release, produces a hydroxyacenaphthoic acid that is decarboxylated to acenaphthenone (V). Monooxygenation of this ketone to the quinone and subsequent hydrolysis generates naphthalene-1,8-dicarboxylic acid (IV), which is further degraded via benzene-1,2,3-tricarboxylic acid (III). A second route involves dioxygenation at C-1 and C-2, followed by dehydrogenation and meta cleavage of the resulting diol. A two-carbon fragment excision of the meta cleavage product yields 9-fluorenone-1-carboxylic acid (II), which appears to undergo angular dioxygenation and further degradation to produce benzene-1,2,3-tricarboxylic acid (III), merging this route with the 7,8-dioxygenation route. Decarboxylation of benzene-1,2,3-tricarboxylic acid to phthalate (VIII), as well as further oxidation of the latter, would connect both routes with the central metabolism. The identification of Z-9-carboxymethylenefluorene-1-carboxylic acid (I) suggests a third route for fluoranthene degradation involving dioxygenation at C-2, C-3, and ortho cleavage. There is no evidence of any further degradation of this compound.

Novel Production of Isochainin by a Strain of Streptomyces sp. Isolated from Rhizosphere Soil of the Indigenous Moroccan Plant Argania Spinosa L

An actinomycete strain (designated Ap1) isolated from the rhizosphere soil of Argania spinosa L. strongly inhibited the growth of two plant pathogens: Fusarium oxysporum f.sp. albedinis and Verticillium dahliae. The spore morphology suggested that the Ap1 strain belonged to the genus Streptomyces. The antifungal compound produced by Ap1 was purified by HPLC and identified as the polyene macrolide, isochainin, by NMR and mass spectroscopy. Ap1 showed normal biosynthesis of isochainin in comparison with S. cellulosae ATTC 12625, in which precursor-directed biosynthesis by feeding ethyl (Z)-16-phenylhexadec-9-enoate to the culture medium is required. In addition, Streptomyces sp. strain Ap1 produces isochainin with a 6.5-fold higher concentration than Streptomyces cellulosae ATTC 12625.

Identification of a novel metabolite in the degradation of pyrene by Mycobacterium sp. strain AP1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons.

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6'-dihydroxy-2,2'-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.

Protein Arp and protein H from group A streptococci. Ig binding and dimerization are regulated by temperature.

Cell surface proteins that bind to the Fc part of Ig are expressed by many strains of group A streptococci, an important human pathogen. Two such bacterial strains, AP4 and AP1, were shown to bind IgA and IgG, respectively, in a temperature-dependent manner. The binding of radiolabeled Ig to the bacterial cells was lower at 37 degrees C than at 22 and 4 degrees C. Similarly, protein Arp, the IgA-binding protein isolated from strain AP4, and protein H, the IgG-binding protein isolated from strain AP1, displayed a strong Ig-binding at 22 degrees C and lower temperatures, and virtually no binding at all at 37 degrees C. The effect was reversible: lowering of the temperature restored the binding and vice versa. A gradual shift between binding and nonbinding took place between 27 and 37 degrees C. Gel chromatography and velocity sedimentation centrifugation showed that protein Arp and protein H appeared as noncovalently associated dimers at 10 and 22 degrees C, and as monomers at 37 degrees C. These results strongly suggest that the dimerization of protein Arp and protein H, rather than the low temperature itself, yielded the strong Ig-binding of the proteins at 10 and 22 degrees C. Indeed, after covalent cross-linking of the dimers at 10 degrees C by incubation with low concentrations of glutaraldehyde, full Ig-binding was achieved even at 37 degrees C. A carboxyl-terminal proteolytic fragment of protein Arp, which completely lacked the IgA-binding capacity at any temperature, showed the same temperature-dependent dimerization as intact protein Arp, suggesting that the Ig-binding part of the protein is not required for dimerization. The implications of these results for the function of Ig-binding group A streptococcal proteins, and their role in the host-parasite relationship are discussed.

Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1

Deletion mutants of Escherichia coli specific for hydrogenase isoenzyme 1 (HYD1) have been constructed and characterized. The hya operon, which contains genes for the two HYD1 structural subunits and four additional genes, was mapped at 22 min on the E. coli chromosome. The total hydrogenase activities of the HYD1-negative mutant and wild-type strains were similar. However, the formate dehydrogenase activity associated with the formate hydrogen lyase pathway was lower in the mutant. The hya mutant (strain AP1), complemented with only the hydrogenase structural genes (hyaAB), produced antigenically identifiable but inactive HYD1 protein. The first five genes of hya (hyaA to hyaE) were required for the synthesis of active HYD1, but wild-type levels of HYD1 activity were restored only when mutant cells were transformed with all six genes of the operon. When AP1 was complemented with hya carried on a high-copy-number plasmid, the HYD1 structural subunits were overexpressed, but the excess protein was unprocessed and localized in the soluble fraction of the cell. The products of hyaDEF are postulated to be involved in the processing of nascent structural subunits (HYAA and HYAB). This processing takes place only after the subunits are inserted into the cell membrane. It is concluded that the biosynthesis of active HYD1 is a complex biochemical process involving the cellular localization and processing of nascent structural subunits, which are in turn dependent on the insertion of nickel into the nascent HYD1 large subunit.

Enhanced gene conversion and postmeiotic segregation in pachytene-arrested Saccharomyces cerevisiae.

Previous study has demonstrated that incubation of yeast cells of strain AP-1 in sporulation medium at 36 degrees permits them to begin meiosis but that they become arrested at pachytene and undergo enhanced intragenic recombination between ade2 heteroalleles. Tetrad analysis was undertaken to characterize the altered program of meiotic recombination more widely. In one set of experiments, pachytene-arrested cells were permitted to resume sporulation upon transfer to the permissive temperature. In the resulting asci, both postmeiotic segregation and gene conversion were increased several-fold at a number of loci relative to unarrested controls, whereas reciprocal recombination increased two- to threefold. Another set of experiments analyzed the genetic consequences of inducing the pachytene-arrested cells to revert directly to mitotic growth without completion of meiosis. The appearance of homozygous sectors from heterozygous markers revealed that these cells had become committed to appreciable recombination but that reciprocal exchange was less frequent than in normal asci. Taken together, the data indicate that pachytene arrest rendered the cells committed to enhanced recombination upon resumption of sporulation but that most of the crossing over did not occur until release from the arrest.--The genetic basis of pachytene arrest by AP-1 was investigated by mating each of its parents with progeny of strain Y55, which is able to sporulate at 36 degrees. Both of these diploids sporulated at 36 degrees, and asci from the one studied further exhibited 2:2 segregation of the sporulation defect, indicating that pachytene arrest is dependent on a recessive, temperature-sensitive allele at a chromosomal locus.

Isolation of genes expressed preferentially during sporulation in the yeast Saccharomyces cerevisiae.

A library of Saccharomyces cerevisiae DNA in the vector lambda Charon 28 was probed for sequences complementary to cDNA made from poly(A)+ RNA isolated from the well-sporulating yeast strain AP1 a/alpha. The RNA was isolated from cells that had been incubated 7, 9, 11, and 13 hr in sporulation medium. DNA complementary to poly(A)+ RNA from alpha/alpha(nonsporulating) AP1 was used as a control, and 46 bacteriophage that gave a stronger response with a/alpha cDNA than with alpha/alpha cDNA were obtained in a screening of three yeast genomes worth of DNA. Two of the bacteriophage appeared to contain a/alpha-specific genes, in that they hybridized to cDNA from vegetative a/alpha RNA. The rest appeared to correspond to a/alpha genes expressed preferentially during sporulation. Restriction endonuclease analysis of four of the cloned sequences revealed a single major region of transcription in each; these regions ranged in size from 2.5 to 4.0 kilobases. RNA blot analysis showed that, in three of the four cases, transcripts of two different sizes were homologous to the cloned sequence. In all four cases, the homologous transcripts appeared at about 7 hr and were decreasing in amount by 13 hr. These results provide evidence for transcriptional control of genes expressed during sporulation and for at least one group of genes that is turned on at about the time of meiosis I in sporulation.