Development and application of a green fluorescent protein sentinel system for identification of RNA interference in Blastomyces dermatitidis illuminates the role of septin in morphogenesis and sporulation.

A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, "X," next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.

Selective expression of the virulence factor BAD1 upon morphogenesis to the pathogenic yeast form of Blastomyces dermatitidis: evidence for transcriptional regulation by a conserved mechanism.

Most dimorphic fungal pathogens grow as non-pathogenic moulds in soil and convert to pathogenic yeast in the host, suggesting that virulence factors are upregulated during phase transition. Such factors have been difficult to identify. We analysed BAD1 (formerly WI-1), a virulence factor in the dimorphic fungus Blastomyces dermatitidis, for expression in yeast and mycelial morphotypes. BAD1 was expressed in yeast but not in mycelia of North American strains of B. dermatitidis, and this expression pattern was confirmed for BAD1 transcript. BAD1 under the control of its promoter was transferred into African B. dermatitidis lacking a native BAD1 locus, and phase-specific expression was conserved. Sequence similarity was identified between the BAD1 promoter and the promoters of two yeast phase-specific genes in Histoplasma capsulatum. In H. capsulatum BAD1 transformants, yeast phase-specific expression of BAD1 was conserved, and no transcript was detected in mycelia. BAD1 beta-galactosidase reporter fusions analysed in B. dermatitidis and H. capsulatum confirmed that BAD1 is transcriptionally regulated in both fungi. BAD1 promoter activity and surface BAD1 expression were detected 6 h after shifting mycelia to 37 degrees C. Thus, BAD1 is expressed after transition to the pathogenic yeast morphotype and is regulated by a mechanism for phase-specific gene expression that appears to be conserved.

The maize brittle 1 gene encodes amyloplast membrane polypeptides.

A chimeric protein, formed of 56 amino acids from the carboxy terminus of the maize (Zea mays L.) wild-type Brittle1 (Bt1) protein fused to the glutathione-S-transferase gene, was synthesized in Escherichia coli, and used to raise antibodies. Following affinity purification, the antibodies recognized a set of 38- to 42-kDa proteins in endosperm from wild-type Bt1 plants, as well as from brittle2, shrunken2 and sugary1 plants, but not in mutant bt1 endosperm. Bt1 proteins were not detected with the preimmune antibodies. A low level of Bt1-specific proteins was detected at 10 d after pollination (DAP) and increased to a plateau at 16 DAP. At the same time, the ratio of slow- to fast-migrating forms of the protein decreased. During endosperm fractionation by differential centrifugation and membrane sedimentation in sucrose gradients, the Bt1 proteins co-purified with the carotenoid-containing plastid membranes. They were localized to amyloplasts by electron-microscopic immunocytochemistry; most of the signal was detected at the plastid periphery. These results are consistent with predictions made from the deduced amino-acid sequence and previous in-vitro experiments that the bt1 locus encodes amyloplast membrane proteins.

Alternative 3' splice acceptor sites modulate enzymic activity in derivative alleles of the maize bronze1-mutable 13 allele.

The defective Suppressor-mutator (dSpm)-induced allele bronze1-mutable 13 (bz1-m13) and many of its derivative alleles are leaky mutants with measurable levels of flavonol O3-glucosyltransferase activity. This activity results from splicing at acceptor site-1, one of two cryptic 3' splice sites within the dSpm insertion in bz1-m13. In this study, splicing in bz1-m13 change-in-state (CS) alleles CS-3 and CS-64 was shown to be altered from bz1-m13; previous work found altered splicing in CS-9. CS-64 is a null allele and lacks the acceptor site-1-spliced transcript because this site is deleted. CS-3 and CS-9 had increased levels of the acceptor site-1 transcript relative to bz1-m13 and increased enzymic activities. A deletion in CS-9 altered splicing by eliminating acceptor site-2. Both acceptor sites were intact in CS-3, but a deletion removed most of a 275-bp GC-rich sequence in dSpm. This suggests that GC-rich sequences affect splicing and is consistent with models postulating a role for AU content in the splicing of plant introns. Splicing does not necessarily occur, however, at the junction of AU-rich intron sequences and GC-rich exon sequences.

Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein.

Based on the protein sequence deduced from a cDNA clone, it has been proposed that the maize bt1 locus encodes an amyloplast membrane metabolite translocator protein (Sullivan, T. D., Strelow, L. I., Illingworth, C. A., Phillips, R. L., and Nelson, O. E., Jr. (1991) Plant Cell 3, 1337-1348). The present work provides further evidence for this hypothesis by showing that the gene product of Bt1 could be imported into chloroplasts in vitro and processed to lower molecular weight mature proteins. More importantly, the imported mature proteins were localized to the inner envelope membrane, where metabolite translocators are located in plastids. In addition, the location of information for targeting to the inner membrane was investigated by constructing and analyzing the import of chimeric precursor proteins. A chimeric protein with the transit peptide of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase fused to the mature region of the Bt1-encoded protein was targeted to the inner envelope membrane of chloroplasts. Moreover, a chimeric protein with the transit peptide of the Bt1-encoded protein fused to the mature protein of the light-harvesting chlorophyll a/b binding protein was targeted to the thylakoid. These results indicate that the transit peptide of the Bt1-encoded protein functions primarily as a stromal targeting sequence. The information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the protein.

Analysis of maize brittle-1 alleles and a defective Suppressor-mutator-induced mutable allele.

A mutant allele of the maize brittle-1 (bt1) locus, brittle-1-mutable (bt1-m), was shown genetically and molecularly to result from the insertion of a defective Suppressor-mutator (dSpm) transposable element. An Spm-hybridizing restriction enzyme fragment, which cosegregates with the bt1-m allele and is absent from wild-type revertants of bt1-m, was identified and cloned. Non-Spm portions of it were used as probes to identify wild-type (Bt1) cDNAs in an endosperm library. The 4.3-kb bt1-m genomic clone contains a 3.3-kb dSpm, which is inserted in an exon and is composed of Spm termini flanking non-Spm sequences. RNA gel blot analyses, using a cloned Bt1 cDNA probe, indicated that Bt1 mRNA is present in the endosperm of developing kernels and is absent from embryo or leaf tissues. Several transcripts are produced by bt1-m. The deduced translation product from a 1.7-kb Bt1 cDNA clone has an apparent plastid transit peptide at its amino terminus and sequence similarity to several mitochondrial inner-envelope translocator proteins, suggesting a possible role in amyloplast membrane transport.

Isolation and characterization of a maize chlorophyll a/b binding protein gene that produces high levels of mRNA in the dark.

A cDNA library prepared using mRNA isolated from red-light irradiated maize seedlings was screened by a difference procedure for clones that represent red-light regulated mRNA. Two such clones were found to represent mRNA for a chlorophyll a/b binding protein (CAB), and one of these (pAB1084) was used to screen a maize genomic library. One positive genomic clone (lambda AB1084) was isolated and sequenced. The gene represented by lambda AB1084, which we designate maize cab-1, contains extensive nucleotide homology within its protein coding region to CAB genes from other species. The boundaries of the transcribed region of the cab-1 gene were determined by S1 nuclease mapping. The 5' terminus of cab-1 mRNA is located 52-54 nucleotides (nt) upstream of the translation start site and 34 nt downstream of a TATA box. As in the case of petunia CAB genes, several poly(A) addition sites are present in mRNA from the cab-1 gene. The 5' flanking DNA of cab-1 contains sequences related to elements that have been implicated in the light-regulated expression of CAB and rbcS genes in other plant systems. Quantitative Northern blot hybridization analysis using a gene specific probe for cab-1 indicates that the mRNA for this gene is present at 0.4% of the total mRNA and up to 80% of the total CAB mRNA in the leaves of dark-grown seedlings. In consequence, although the degree of up-regulation by white light is only moderate (3- to 6-fold), cab-1 transcripts account for approximately 2% of the mRNA in the leaves of light-grown seedlings.

Tissue-specific effects of maize bronze gene promoter mutations induced by Ds1 insertion and excision.

Bz-wm is an allele of the Bz locus of maize isolated by McClintock (1962) as a derivative of bz-m2. It contains a Ds1 insertion 63 bp upstream of the start of transcription and a 3 bp insertion in the coding region at the site of the Ac element that was present in bz-m2. Bz-wm produces, in the aleurone layer of the endosperm, low amounts (approximately 1% of wild-type) of a Bz-gene encoded UDP-glucose: flavoid 3-0-glucosyltransferase (UFGT) polypeptide with altered thermal stability. Three phenotypically wild-type derivatives, Bz' (wm)-1, Bz' (wm)-2 and Bz' (wm)-3, were isolated in the presence of Ac and shown to have excised the Ds1 element but not fully restored UFGT activity in endosperm assays. In the studies reported here, we have further analyzed these Bz' derivatives of Bz-wm by determining the DNA sequences left behind on Ds1 excision, and by measuring the amount of UFGT activity and/or Bz mRNA conditioned by Bz-wm and the Bz' derivatives in different tissues. The data indicate that tissue-specific differences in expression of the Bz gene have been produced in alleles with mutations caused by transposable elements Ac and Ds. These mutations may affect either the amount of Bz transcription or the stability of the UFGT polypeptide. The sequence or spacing in the -63 region of the Bz promoter appears to be critical for maximum expression in aleurone and husk but not in pollen and pigmented seedling tissue.

Neisseria meningitidis bacteremia in children: quantitation of bacteremia and spontaneous clinical recovery without antibiotic therapy.

The relationship between the magnitude of bacteremia due to Neisseria meningitidis and the clinical diagnosis was determined for 43 children who had fever in the presence or absence of focal signs of infection. Bacteremia was quantitated by the previously described procedure using heparinized blood (0.2 to 1.0 mL). Additionally, blood was cultured by means of the radiometric Bactec technique. Seventeen patients had meningitis, 12 had meningococcemia, 13 had unsuspected or "occult" bacteremia, and five had other diagnoses. "Occult" bacteremia was diagnosed initially in four patients, but subsequently meningitis was diagnosed. All 13 patients with 500 or more organisms per milliliter had meningitis or meningococcemia in contrast to 12 (55%) of 22 patients with less than 500 organisms per milliliter (P less than or equal to .0035). Only 18 (42%) of these patients bacteremic with N meningitidis presented with petechiae or purpura. All 13 children with occult bacteremia were sent home after blood cultures were obtained; six of the 13 received a regimen of oral amoxicillin for otitis media. At reexamination (interval 16 to 119 hours) four had meningitis, seven were clinically improved (afebrile, negative blood culture, without invasive disease), and two were still mildly febrile with negative blood culture. Three of these bacteremic children experienced spontaneous clinical and bacteriologic resolution without antibiotic treatment. This has not been previously reported.

Half-lives of beta and gamma globin messenger RNAs and of protein synthetic capacity in cultured human reticulocytes.

The turnover rates of beta and gamma globin messenger RNAs and of beta and gamma globin protein synthesis in human reticulocytes have been measured. Our goal was to determine whether beta globin mRNA is significantly more stable than gamma globin mRNA during the final stages of erythroid cell maturation. Such a result could explain the reported increase in the beta-gamma protein synthetic ratio during erythroid maturation. As determined by molecular hybridization and cell-free translation assays, the half-lives of both mRNAs are 20 to 29 hours in adult and neonatal reticulocytes. Protein synthetic capacity in intact cells decays with a half-life of six to eight hours, but beta protein synthesis declines at the same rate as gamma. Therefore, the changing ratio of fetal to adult hemoglobin synthesis during late erythroid maturation does not result from differences in mRNA turnover rates or changes in translation efficiencies. These data, coupled with those obtained with immature erythroid cells (Farquhar et al, Dev Biol 85: 403, 1981), suggest that, during erythroid maturation, the gamma-beta globin protein synthesis ratio declines because gamma gene transcription ceases earlier than beta gene transcription. Our results also indicate that the protein synthetic machinery, not the quantity of mRNA, becomes rate-limiting for globin production in cultured reticulocytes.

Advantages of BACTEC hypertonic culture medium for detection of Haemophilus influenzae bacteremia in children.

The hypertonic and aerobic culture media in the BACTEC system were compared for the detection of Haemophilus influenzae bacteremia in children. Of 1,611 blood cultures, 30 were positive for this pathogen. The aerobic and hypertonic media gave positive results in 28 and 29 cultures, respectively. Within the first 12 h, H. influenzae was detected in the hypertonic medium in 48.5% of the positive cultures as compared to 35% for the aerobic medium. Importantly, after the first 12 h, the hypertonic medium yielded positive results sooner than did the aerobic medium, the difference being statistically significant (P less than 0.01). The hypertonic medium yielded positive results earlier than the aerobic medium in nine cultures; the reverse was seen in only one culture. Furthermore, the aerobic medium gave negative growth index readings despite growth, as shown by microscopy and subculture, in 43% of the total cultures in contrast to only 13% for the hypertonic medium, a significant difference (P less than 0.05). Thus, the present study indicates a distinct advantage of hypertonic medium compared with aerobic medium in the automated BACTEC system for earlier detection of H. influenzae bacteremia and is recommended for those age groups in which this pathogen plays a predominant role.

Relationship between the magnitude of bacteremia in children and the clinical disease.

The relationship between the magnitude of bacteremia due to Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis and the clinical diagnosis was determined on 79 children who were not receiving prior antibiotic therapy and had fever, either in the presence or absence of focal signs of infection. Bacteremia was quantitated by the recently described Quantitative Direct Plating procedure in which heparinized blood (0.5 ml each) is plated onto blood and chocolate agar plates. Additionally, blood was cultured by means of the radiometric Bactec technique. In the case of H. influenzae and S pneumoniae, 23 (92%) of 25 patients with more than 100 organisms per milliliter of blood had meningitis or epiglottitis in contrast to only four (9.5%) of 42 patients with less than 100 organisms (P less than .001). No significant difference was noted in the magnitude of bacteremia due to N meningitidis among 12 patients with meningitis or other serious infections. The possible predictive value of the quantitation of bacteremia is illustrated by the observation of three children with seemingly mild respiratory infection and counts in excess of 100 organisms per milliliter who, within 20 hours, developed meningitis or epiglottitis. High bacterial counts of H influenzae and S pneumoniae in excess of 100 organisms per milliliter of blood should alert the physician to the existence or possible development of serious disease.

Diagnosis of bacteremia in children by quantitative direct plating and a radiometric procedure.

During a 1-year period, three bacteriological systems for detecting bacteremia in children were analyzed, namely, the BACTEC system (Johnston Laboratories, Inc., Cockeysville, Md.), the Fisher/Lederle bottle (Lederle Diagnostics, Pearl River, N.Y.), and a direct plating method of blood termed quantitative direct plating (QDP). Of 2,123 blood cultures, 135 (6.4%) were positive; Haemophilus influenzae type b, Neisseria meningitidis, and Streptococcus pneumoniae accounted for 3.4%, representing 61 patients, other pathogens accounted for 0.6%, and contaminants accounted for 2.4%. Of 72 cultures yielding H. influenzae, N. meningitidis, and S. pneumoniae, 60 were recovered by both broth systems, 2 by BACTEC only and 10 by Fisher/Lederle bottle only. The BACTEC system failed to register a positive growth index reading by 24 h in 15 cultures which were positive for H. influenzae, even though growth had occurred, as shown by positive subculture and microscopy at this time. QDP detected 89% of the cultures positive for H. influenzae and N. meningitidis, of which 55% yielded results before either broth procedure. Only 50% of the cultures positive for S. pneumoniae yielded growth on QDP. This difference in the recovery rate probably is accounted for by the number of organisms in the blood. Thus, more than 100 organisms per ml of blood were found in 71% of cultures positive for H. influenzae and N. meningitidis but in only 7% of those positive for S. pneumoniae. These studies, then, have revealed that H. influenzae, which grew well in BACTEC broth, did not, however, give a significant growth index reading during day 1 of incubation, in contrast to N. meningitidis and S. pneumoniae. The QDP system not only provided information on the magnitude of bacteremia due to H. influenzae and N. meningitidis but frequently allowed earlier diagnosis and, thus, proved to be a valuable, simple, and inexpensive supplementary technique for broth cultures, although not for the diagnosis of S. pneumoniae bacteremia.

Spinal cord involvement in acute bacterial meningitis.

A 25-month-old boy had the development of respiratory arrest and quadriplegia with a T-10 sensory level during the acute phase of Haemophilus influenzae meningitis. The sequelae of spinal cord involvement of bacterial meningitis are reviewed. A possible mechanism of the spinal cord involvement in this case is discussed with reference to known pathology of H influenzae meningitis.

The effects of ampicillin and trimethoprim/sulfamethoxazole on the periurethral flora of children with urinary tract infection.

Ampicillin and trimethoprim/sulfamethoxazole were shown to be of similar efficacy in the treatment of acute urinary tract infection of children. It was of interest to determine the effects of these antimicrobial drugs on the periurethral flora and recurrence rates. To this end, seventeen girls with twenty-two separate infections of the urinary tract were treated randomly with a ten-day course of either ampicillin or trimethoprim/sulfamethoxazole. Cultures of the urine and periurethral area were obtained before, during (third day), and after (seventeenth day) therapy. All Escherichia coli strains were serotyped. Both treatments resulted in the disappearance of the pathogens from the urine by the third day in all cases, and in all but one patient on the seventeenth day. The causative agents persisted more frequently in the periurethral area than in the urine on both the third and seventeenth days in patients treated with either ampicillin or trimethoprim/sulfamethoxazole. The recurrence rates by the seventeenth day were 50% (4/8) in the ampicillin group, and 14% (2/14) in the trimethoprim/sulfamethoxazole group. Although suggestive in favor of the latter treatment, the difference is not statistically significant. In two of the three re-infections in the ampicillin group the microorganisms causing the second attack were present in the periurethral area on the third day. Sixteen of the seventeen girls were studied radiologically; six (37%) had radiologic abnormalities.

Iron toxicity screening.

Fischer's method for rapid detection of acute iron toxicity is modified to suit pediatric cases. TPTZ (2,4,6-tripyridyl-s-triazine) is the chromogen of choice since in a small volume of serum slight to moderate hemolysis can cause a false positive result bathophenanthroline. Ordinary labware is amenable to this simplified procedure.