David Rosenthal's Abstracts

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1993 Abstracts

1. POLYMERASE CHAIN REACTION (PCR) DETECTION OF DNA FOR THE ALTERNARIA ALLERGEN ALT A1. J Portnoy MD, D Rosenthal, S Horner DO, F Pacheco MS, J Landuyt BS, C Barnes PhD. Kansas City, Missouri.
Rapid amplification of cDNA ends (RACE) is used to generate full length cDNA copies of mRNA transcripts. It uses PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Reverse transcription and PCR are used for the single sided transcript. An oligonucleotide generated from the known N-terminal amino acid sequence along with an oligo-dT primer is used to generate the first strand reverse transcript for 5' transcripts, resulting in full length extension.
Immunoblot experiments of Alternaria (ALT) allergens such as Alt a1 have raised questions that can only be answered by identification of its DNA sequence in several different strains. To initiate this process, the following experiments were per-ormed.
ALT was grown on defined medium in a fermenter with constant stir-ing and aeration for 5 days. Rapidly expanding mycelia were removed, frozen and then ground in liquid nitrogen, and disintegrated in a high speed homogenizer. The product was denatured in guanidine HCl with RNase inhibitors and extracted using a phenol chloroform method. mRNA was further isolated using oligo dT-linked magnetic beads. Purified mRNA was reacted with reverse transcriptase and 0.45 Ķg of the first copy ssDNA was subjected to PCR. The first strand oligo:
5'-CCC GTG ACC ACC CAG GGC GAC TAC GTG TCC AAG ATC-3' 
    P   V   T   T   Q   G   D   Y   V   S   K   I 
was synthesized from the known N-terminal amino acid sequence of Alt a1 and the second strand oligo was the 28 mer 5'-CAA TTC GCG GCC GC(T)15-3'. PCR was carried out in a thermal cycler alternating between 1 minute at 94•C, 1 minute at 50•C, and 2 minutes at 72•C. Amplified DNA was analyzed by standard electrophoretic methods in a 3% agarose gel, stained with ethidium bromide and visualized on a transilluminator.
The results indicate that mRNA for the Alt a 1 allergen is present in the mycelia of this strain of Alternaria. The electrophoretic analysis of the PCR product DNA indicates that there are two products of the reaction. One transcript is 375 base pairs and the second is 285 base pairs. These would correspond to 15KD and 12KD peptides which is approximately the reported size for the reduced form of Alt a1.

2. PRODUCTION OF RECOMBINANT ALTERNARIA ALLERGENS.D Rosenthal, J Portnoy MD, S Horner DO, F Pacheco MS, J Landuyt BS, C Barnes PhD. Kansas City, Missouri.
The variability of extracts derived from Alternaria (ALT) is well documented. One possible solution to this problem might be the production of recombinant allergenic proteins in E. coli by inserting DNA taken from ALT which is specific for production of a particular allergen. To explore this possibility, the following experiments were performed.
A strain of ALT was grown on minimum salts and glucose in a fermentation container with constant stirring and aeration for 5 days. Rapidly expanding mycelia were removed from the culture and frozen in liquid nitrogen. Frozen mycelia were ground in liquid nitrogen and disrupted with short bursts in a high speed homogenizer. Homogenized mycelia were denatured in guanidine thiocynate in the presence of RNase inhibitors and extracted using a phenol chloroform method. Purified RNA was reacted with reverse transcriptase and an aliquot of first copy single strand DNA was enriched for the presence of DNA coding for an Alternaria allergen by PCR amplification.
Amplified DNA as well as whole cDNA was modified to contain a EcoR1 restriction site on one side and a Not1 site on the other end. Modified DNA was then spliced into lambda gt11 phage. The packing reaction yielded recombinant libraries with phage titers in the range of 10E5 plaque forming units. Amplified libraries were screened for the presence of allergenic proteins with monoclonal antibodies (MAb) directed to ALT allergens and with IgE containing human sera from ALT sensitive patients. Positive plaques were cloned and used in the production of E. coli lysogens. Positive colonies were chosen and grown under conditions specific for the production of the recombinant protein. Expressed proteins were tested using a MAb based sandwich ELISA for allergenic proteins from Alternaria and by SDS-PAGE immunoblotting analysis.
Further analysis of these recombinant proteins should permit a better understanding of ALT allergens.

3. STRUCTURAL DETERMINATION OF A HUMAN IGE RECOGNIZED PROTEIN FROM ALTERNARIA ALTERNATA. C.Barnes, D.Rosenthal, F.Pacheco, J.Landuyt and J.Portnoy. Children’a Mercy Hospital and UMKC, Medical School, Kansas City, MO 64108
IgE recognized proteins derived from environmental sources cause significant human morbidity and mortality. Elucidation of structural characteristics of these proteins contributes to improved diagnosis and treatment of asthma and allergy. To study the structure of a 31kDa allergenic protein identified in the mold, Alternaria alternata (ALT), the following experiments were performed.
ALT was grown in a fermenter with constant stirring and aeration. Monoclonal antibodies (MAB’s) against the 31kDa protein were produced from electrophoretically separated material. Additional protein was purified by MAB affinity chromatography. For RNA purification, rapidly expanding mycelia was ground in liquid nitrogen and extracted using a phenol chloroform method. RNA was reacted with reverse transcriptase and first copy ssDNA was amplified by PCR. Amplified DNA as well as ds-cDNA was inserted directionally into phage and a cDNA library was produced. The library was screened with MAB’s and IgE containing human sera from ALT sensitive patients. Positive clones were analyzed by PCR and sequenced. PCR analysis of cDNA using an oligonucleotide derived from the N-terminal sequence and oligo dT indicates a nucleotide of 295 base pairs. Sequence analysis of the PCR product yields a reading frame containing 66 amino acids. A computer search for this sequence found no homologous proteins. Northern blotting analysis indicates the PCR product to be the C-terminal third of a 1000 base mRNA segment.

1994 Abstracts

4. IDENTIFICATION OF THE ALT AI ALLERGEN IN EIGHT STRAINS OF ALTERNARIA. D Rosenthal, R Crenshaw MS, R Esch PhD, F Pacheco MS, J Landuyt BS, C Barnes PhD, J Portnoy MD. Kansas City, Missouri and Lenoir, North Carolina.
The variability of extracts derived from Alternaria (ALT) is well documented. At the protein level significant allergens are often absent or in very low concentrations. To to determine if these variations occur at the DNA level, the following experiments were performed. Eight strains of ALT obtained from 4 different research groups were grown on minimum salts and glucose in a fermentation container with constant stirring and aeration for 5 days. Rapidly expanding mycelia were removed from the culture and frozen in liquid nitrogen. Frozen mycelia were ground in liquid nitrogen and disrupted with short bursts in a high speed homogenizer. Homogenized mycelia were denatured in guanidine thiocynate in the presence of RNase inhibitors and extracted using a phenol chloroform method. After quantitative measurements were made by UV absorption, alliquots containing equal amounts of RNA were applied to a denaturing agarose gel, electrophoresed and blotted by capillary action onto nitrocellulose. RNA blots were probed with a radiolabeled 300 BP DNA fragment which codes for the N-terminal portion of the Alt aI allergen. Probed blots were visualized on a phosphorimager.
Analysis of the RNA blot showed a single band at about 1000 bases in each of the eight strains of Alternaria tested. Densitometric analysis of the blots obtained indicated that some strains contained as much as 10 times the mRNA for this allergen as other strians. The results of the northern blot analysis is compared with quantitative ELISA analysis for the protein allergen in these same strains. These data suggest that quantitative differences in allergen production among differing strains of Alternaria occur not only at the protein level but also at the level of the genetic material. Post translational processing of proteins is an important contributor to the complexity of ALT extracts and that such processing must be accounted for when standardization is attempted.

5. PARTIAL AMINO ACID SEQUENCE FOR THE ALT AI ALLERGEN. J Portnoy MD,F Pacheco MS, D Rosenthal,J Landuyt BS, C Barnes PhD. Kansas City, Missouri.
Sequencing of IgE recognized proteins either at the amino acid or DNA level has been conducted for many significant allergens. Elucidation of structural characteristics of these proteins contributes to improved diagnosis and treatment of asthma and allergy. To study the structure of a 31kD allergenic protein identified in the mold, Alternaria alternata(ALT), the following experiments were performed.
ALT was grown in a fermenter with constant stirring and aeration. Monoclonal antibodies (MAB's) against the 31kD protein were produced from electrophoretically separated material. Additional protein was purified by MAB affinity chromatography. For RNA purification, rapidly expanding mycelia were ground in liquid nitrogen and extracted using a phenol chloroform method. RNA was reacted with reverse transcriptase and first copy ssDNA was amplified by PCR. Amplified DNA as well as ds-cDNA was inserted directionally into phage and a cDNA library was produced. The library was screened with MAB’s and IgE containing human sera from ALT sensitive patients.
PCR analysis of cDNA using an oligonucleotide derived from the N-terminal sequence and oligo dT indicates a nucleotide of 295 base pairs. Sequence analysis of the PCR product yields a reading frame containing 85 amino acids. A computer search for this sequence found no homologous proteins. Northern blotting analysis indicates the PCR product to be the C-terminal third of a 1000 base mRNA segment.

6. PRODUCTION OF A FUSION PROTEIN CONTAINING RECOMBINANT ALT AI, A MAJOR ALTERNARIA ALLERGEN. F Pacheco MS, D Rosenthal, J Portnoy MD,, J Landuyt BS, C Barnes PhD. Kansas City, Missouri.
The allergenic composition of Alternaria (ALT) extracts is extremely variable. This makes it difficult to characterize specific allergenic glycoproteins. One solution to this may be with the production of recombinant allergenic proteins. By inserting DNA coding for a known ALT allergen into an appropriate vector, it is possible to produce usable quantities of protein, To this end, the following experiments were performed.
A strain of ALT was grown on minimum salts and glucose in a fermenter with constant stirring and aeration for 5 days. Rapidly expanding mycelia were removed from the culture and frozen in liquid nitrogen. These were next ground in liquid nitrogen and RNA was extracted using a phenol chloroform method. RNA was reacted with reverse transcriptase and an aliquot of first copy single strand DNA was enriched for the presence of cDNA coding for an Alternaria allergen by PCR amplification. cDNA libraries were screened for the presence the desired insert by PCR and for allergenic proteins with monoclonal antibodies (MAb) directed to ALT allergens and with IgE containing human sera from ALT-sensitive patients. Positive plaques were cloned and used in the production of the desired DNA. Specific oligonucleotide primers were designed to give a PCR product containing a full copy of the desired DNA along with the necessary restriction sites to allow insertion into a plasmid specific for expresson of protein.
Colonies were screened for expressed proteins by SDS-PAGE and positive clones were sequenced. The product was fused to the C-terminus of glutathione sulfhydryl transferase which aided in purification and could be easily removed enzymatically. Expressed proteins were tested using a MAb-based sandwich ELISA for allergenic proteins from Alternaria and by SDS-PAGE immunoblotting analysis. Further analysis of this product should clarify many of the properties of Alt aI

7. STUDIES ON THE MRNA FOR ALTERNARIA ALLERGEN ALT AI BY POLYMERASE CHAIN REACTION (PCR). C Barnes PhD , D Rosenthal, F Pacheco MS, J Landuyt BS, J Portnoy MD. Kansas City, Missouri.
Traditional protein isolation and purification methods including immunoblotting have yielded less than a consensus among researchers about the nature of the allergens found in Alternaria (ALT) extracts. Because of the apparent differences in allergens at the protein level, efforts to characterize allergen content in differing strains of ALT must concentrate on the level of the nucleotides. PCR is a powerful tool for the analysis of native DNA and cDNA inserts cloned into E. coli through the use of phage or plasmid vectors. To characterize ALT allergen content at the DNA and mRNA levels, the following studies were conducted.
ALT was grown on defined medium in a fermenter with constant stirring and aeration for 5 days. Rapidly expanding mycelia were removed, frozen and then ground in liquid nitrogen, and disintegrated in a high speed homogenizer. The product was denatured in guanidine HCl with RNase inhibitors and extracted using a phenol chloroform method. RNA was reacted with reverse transcriptase. DNA was then spliced directionally into lambda gt11 phage. Amplified libraries were screened for the presence of DNA coding for allergenic proteins with PCR using synthetic oligonucleotides specific for a 150 base pair region of Alt aI. Positive plaques were then screened for size using PCR with oligonucleotides residing on either side of the insert region.
PCR screening of cDNA libraries indicates that mRNA coding for Alt a1 is as large as 600 base pairs. MRNA and DNA specific for the Alt aI allergen can be identified from several strains of ALT using PCR techniques.

1995 Abstracts

8. PRODUCTION OF A RECOMBINANT PROTEIN FROM ALTERNARIA CONTAINING THE REPORTED N-TERMINAL OF THE ALT A1 ALLERGEN. CS Barnes, F Pacheco, J Landuyt, D Rosenthal, F Hu , J Portnoy . Section of Allergy/Immunology, The Children's Mercy Hospital, Kansas City, MO.
Allergen content of extract derived from Alternaria is somewhat variable. Allergenic molecules from Alternaria that appear as differing molecular size bands on IgE probed immunoblots may have a great deal of sequence homology and differ only in the length of the amino acid chain. One method to study this problem is to produce recombinant proteins from Alternaria. To explore these possibilities, the following experiments were performed.
A strain of Alternaria was grown on minimum salts and glucose in a fermentation container with constant stirring and aeration. Rapidly expanding mycelia were removed from the culture and mRNA was extracted. Purified mRNA was enriched for the presence of DNA coding for an Alternaria allergen by PCR amplification. Modified DNA was then spliced into lambda gt11 phage and yielded a recombinant library with 10E5 PFU. The library was screened for the presence of allergenic proteins using IgE containing human sera from Alternaria-sensitive patients. Positive plaques were cloned.
PCR analysis of positive clones using an oligonucleotide from the reported N-terminal sequence of Alt a1 indicated an insert of 295 base pairs. Sequence analysis yielded a reading frame containing 84 amino acids and confirmed that this segment contained the code for the reported N- terminal amino acid sequence of Alt a1. A computer search for this sequence found no homologous proteins in the Entrez® sequences. Northern blotting studies on RNA purified from nine strains of Alternaria with the radiolabeled 274 BP DNA fragment indicated that this sequence was present in all strains. The 274 BP nucleotide was spliced into the Pflag® vector and clones containing insert in the proper reading frame were identified. The presence of recombinant protein in the clones was verified by SDSPAGE time studies. Protein produced in time studies was shown by immunoblotting and sandwich EIA to bind human IgE from Alternaria sensitive patients. This recombinant protein, containing amino acid sequence for Alt a1, is bound by human IgE and therefore should be useful as a model for studying allergy to the native Alternaria glycoprotein. Further research should define where this sequence occurs in the Alternaria genome and should determine the sequence of the entire protein.

9. NORTHERN BLOTTING STUDIES ON ALT AI MRNA AND 5.8S RRNA IN NINE STRAINS OF ALTERNARIA. D Rosenthal, R Crenshaw MS, R Esch PhD, F Pacheco MS, J Landuyt BS, F Hu DVM, C Barnes PhD, J Portnoy MD. Kansas City, Missouri and Lenoir, North Carolina.
Allergen content of extracts derived from Alternaria (ALT) varies greatly. To determine if similar variations are present in mRNA we performed the following experiments.
For 9 strains of ALT, rapidly expanding mycelia were frozen at -70°C, homogenized, denatured in guanidium thiocynate in the presence of RNase inhibitors, and extracted using a phenol chloroform method. Aliquots containing equal amounts of RNA were applied to a formaldehyde denaturing agarose gel, electrophoresed, and blotted by capillary action onto nylon66 membrane. RNA blots were probed with a radiolabeled 274 BP DNA fragment which codes for the reported N-terminal portion of Alt aI and with a 156 BP DNA fragment from 5.8S ribosomal RNA. Spent culture filtrate and extracted cellular material were assayed for the presence of Alt a1 by sandwich EIA.
When normalized using the 5.8S rRNA, Alt a1 mRNA in the 9 strains varied as much as 4-fold. Alt a1 protein correlated only moderately (.56, p=0.1) with the amount of mRNA. This correlation was much lower (.10) for another ALT allergen, GP70.
The observed low correlation between mRNA and allergen levels may be caused by protein degradation or immunologically cross reactive proteins.

10. IGE BINDING TO A SYNTHETIC RECOMBINANT PEPTIDE CONTAINING THE N- TERMINAL AA SEQUENCE OF ALT A1. F Hu DVM , F Pacheco MS, J Landuyt BS, D Rosenthal, C Barnes PhD,.J Portnoy MD. Kansas City, Missouri.
The characteristics of Alternaria (ALT) allergens may be studied via production of recombinant allergenic proteins. Inserting DNA coding for a known ALT allergen into an appropriate vector allows the production of usable quantities of the relevant allergen devoid of carbohydrate. The IgE binding characteristics of this allergen devoid of its carbohydrate moity will provide a great deal of insight into the nature of the allergen. To begin this process of discovery, the following experiments were performed. A strain of ALT was grown on minimum salts and glucose in a fermenter with constant stirring and aeration for 5 days. Rapidly expanding mycelia were removed from the culture and used to produce a cDNA library for Alternaria.The library was screened for allergenic proteins with monoclonal antibodies (MAb) directed to ALT allergens and with IgE containing human sera from ALT-sensitive patients.A clone of E. coli containing 300 BP segment of Alternaria DNA was identified. Sequence analysis determined that this segment contained the code for the known N- terminal amino acid sequence of the Alt a1 allergen. The segment was spliced into the Pflag® vector for protein production and 4 clones containing the insert in the proper reading frame were identified. These clones were then induced to produce the recombinant protein. Recombinant proteins produced were purified by affinity chromatography.
Expressed proteins were tested for IgE binding by SDS-PAGE immunoblotting analysis using sera from Alt sensitive patients and by three ELISA based methods. Further analysis of this product should clarify many of the properties of Alt aI.

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