Recombinant cDNA Library Research Articles

A Tarantula-Venom Peptide Antagonizes the TRPA1 Nociceptor Ion Channel by Binding to the S1–S4 Gating Domain

The venoms of predators have been an excellent source of diverse highly specific peptides targeting ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1). We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by coexpression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. We constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site. These studies establish our approach, which we term "toxineering," as a generally applicable method for isolation of novel ion channel modifiers and design of ion channel modifiers with altered specificity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing biophysical mechanisms of TRPA1 gating and the physiology of TRPA1 function in nociceptors, as well as for potential clinical application in the context of pain and inflammation.

Identification of Four New HLA Class II Restricted Minor Histocompatibility Antigens Contributing to Graft Versus Leukemia Reactivity.

Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+ and CD4+ T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+ T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+ T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+ T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+ CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+ T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+ T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+ T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.

Humoral immune responses against tumor‐associated antigen OVA66 originally defined by serological analysis of recombinant cDNA expression libraries and its potentiality in cellular immunity

Immunotherapy for cancer relies on the identification of tumor antigens and efficacy of antitumor immune responses. Serological analysis of recombinant cDNA libraries (SEREX), which is based on the spontaneous humoral responses against potential tumor antigens, has provided a novel strategy for searching novel tumor-associated candidates. Through SEREX analysis, we have identified 24 distinct gene clones by immunoscreening of a cDNA library derived from an ovarian cancer patient. Among these genes, a novel gene, OVA66, was found to be expressed significantly higher in carcinoma samples from cancer patients than in normal controls. Comparing humoral responses to OVA66 between tumor patients and healthy donors, it has been shown that the IgG level against OVA66 was significantly elevated in the serum of cancer patients from different histological types of cancer. To determine whether SEREX-defined OVA66 can trigger promising cytotoxic T lymphocyte (CTL) responses, human leukocyte antigen (HLA)-A*0201-restricted T-cell epitopes were predicted through a computational algorithm. Of four predicted peptides, p306-314 (L235) possesses the ability to induce efficient peripheral blood lymphocyte (PBL)-derived CTL responses capable of specifically recognizing peptide-pulsed T2 cells and lysing carcinoma cell lines expressing both HLA-A2 and OVA66 as determined by cytotoxicity and enzyme-linked immunospot assay (ELISPOT). Taken together, our results demonstrate that the SEREX-defined tumor-associated antigen OVA66 can elicit humoral immunity and may also serve as a potential candidate for T-cell-based immunotherapy for cancer.

Identification of phosphatidylinositol 4-kinase type II β as HLA class II-restricted target in graft versus leukemia reactivity

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because HLA class II molecules are predominantly expressed on hematopoietic cells, mHag-specific CD4(+) T cells may selectively mediate graft versus leukemia (GvL) reactivity without graft versus host disease (GvHD). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQB1*0603)-restricted mHag LB-PI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II beta gene. A polyclonal CD4(+) T cell response against LB-PI4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alloSCT. LB-PI4K2B-1S-specific CD4(+) T cells recognized and lysed the CD34(+) CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-gamma and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific CD4(+) T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating CD8(+) T cell immunity as helper cells.

Identification and characterization of a novel gene encoding a SEREX antigen in chronic myeloid leukaemia.

The novel gene encoding Clone 4, previously isolated through the Serological screening of recombinant expression cDNA library (SEREX) in chronic myeloid leukaemia (CML), was characterized. It is localized to chromosome 7 at q22, occupies approximately 4.5 kb of DNA and comprises 4 exons. Three single nucleotide polymorphisms and a 13 base deletion/insertion polymorphism have been identified. The mRNA transcript was approximately 2.4 kb and contained a long 5'-untranslated region of almost 1 kb before the candidate open reading frame. Northern blot analysis and multiple tissue expression array indicated a restricted normal tissue distribution. Our findings provide a platform for future investigations into the role of the gene product in CML.

Full-length-enriched cDNA libraries from Echinococcus granulosus contain separate populations of oligo-capped and trans-spliced transcripts and a high level of predicted signal peptide sequences

The tissue-dwelling larval stages of the cestode Echinococcus granulosus are intimately associated with the host, implying that a range of molecular mediators may be secreted by the parasite into the host environment. These mediators are being sought through a transcriptome-based analysis, using recombinant cDNA libraries. Conventional cDNA libraries of E. granulosus contain high levels of mitochondrial transcripts, as well as host (bovine) genomic DNA. In particular, 60% of a conventional protoscolex stage cDNA library corresponds to the large subunit (LSU) of mitochondrial rRNA. We attribute the presence of LSU rRNA copies to its polyadenylation in E. granulosus. To circumvent this problem, we adapted the 5′ Rapid Amplification of cDNA Ends (RNA-ligase mediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5′ end of full-length mRNA. By ligating a specific oligonucleotide (oligo-cap) to 7MG-bearing mRNA, three cDNA libraries were made by PCR from oligo-cap and oligo-dT primers. Analysis of these libraries showed that mitochondrial RNA contaminants had been excluded. Moreover, no bovine genomic sequences were detected. In parallel, we constructed three cDNA libraries using the newly described trans-spliced leader (SL) from Echinococcus. Although these represent a smaller subset of parasite genes, mitochondrial and genomic contributions were again excluded. In both cases, a majority of cDNAs (61–92%) were judged to contain the initiation ATG codon, and 11–27% of inserts included potential N-terminal signal sequences. The 5′ UTR tracts of most oligo-capped cDNAs were <100 nt, although ∼8% were longer than this. Among the trans-spliced cDNAs, 43% potentially utilise the AUG donated by the SL, and in only 6% was the SL separated from an endogenous putative start site by >60 nt. Sequence analysis of randomly selected clones shows virtually no overlap between the oligo-capped and SL libraries, indicating that trans-spliced E. granulosus mRNAs appear to be insensitive to the enzymatic treatments used to ‘oligo-cap’ unspliced mRNAs. The oligo-capped and SL strategies represent efficient and complementary pathways to isolate full-length cDNA clones from this cestode parasite and, possibly, from related parasitic flatworms.

The progressing clinical utility of DNA microarrays.

The progressing clinical utility of DNA microarrays.

Molecular analysis of skewed Tcra-V gene use in T-cell receptor β-chain transgenic mice

The influence of beta-chain diversity on the expressed T-cell receptor (TCR) alpha-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR beta-chain gene. Analysis of these mice using alpha-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded beta chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards alpha chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in beta-transgenic mice is exhibited almost exclusively by CD4+ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8+ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed beta chains can profoundly influence the selection of companion alpha chains expressed in the periphery, and that alpha-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3alpha domain participates in direct contact with the MHC.

Epitope Mapping of Inhibitory Monoclonal Antibodies to Human von Willebrand Factor by Using Recombinant cDNA Libraries

Two recombinant expression libraries containing small (300-600 base pairs) cDNA fragments of von Willebrand Factor (vWF) were screened in order to map the epitope of monoclonal antibodies (MAbs) to vWF. Among eleven MAbs tested, seven were effectively mapped. The epitopes of MAbs 418 and 522, which inhibit the binding of vWF to Factor VIII (FVIII), were localized between Leu 2 and Arg 53 and between Glu 35 and Ile 81 of the vWF subunit respectively, within the N-terminal trypsin fragment called SpIII-T4 [amino acids (aa) 1-272] which contains a binding domain for FVIII. The epitope of MAb 710, which inhibits the binding of vWF to glycoprotein Ib (GPIb), was identified between Ser 593 and Ser 678 on the tryptic 52/48 kDa fragment (aa 449-728) which contains binding domains for GPIb, collagen, heparin, sulfatides and subendothelium extracellular matrices. The epitope of MAb 723, which does not interfere with any known function of vWF, was localized between Ser 523 and Gly 588. The epitopes of MAb 505 and MAb 400, which inhibit the binding of vWF to collagen, were identified between Leu 927 and Arg 1114 within the SPI fragment (aa 911-1365) corresponding to the central part of the vWF subunit. The epitope of MAb 9, which inhibits the binding of vWF to GPIIb/IIIa, was identified in the C-terminal part of the vWF subunit between Gln 1704 and Asp 1746, the latter being the third aa of the RGD sequence common to adhesive proteins and serving as a recognition site for integrin receptors.

Cloning of a Recombinant Complementary DNA to a Baboon (Papio anubis) Estradiol-Dependent Oviduct-Specific Glycoprotein

The estradiol-dominated baboon oviduct synthesizes and secretes a glycoprotein (mol wt, 120,000) that binds to oocyte zona pellucida in vivo. This glycoprotein separates into two major isoelectric variants (pl 8.0 and 4.5) on two-dimensional electrophoretic gels. This study was undertaken to further characterize the steroid-controlled gene expression of this glycoprotein. A recombinant cDNA library was prepared to poly(A)+ RNA isolated from oviducts obtained from estradiol-treated baboons. The library was screened with a polyclonal antibody prepared against the acidic component (pl 4.5) of the glycoprotein. A single positive plaque was purified. Digestion of the recombinant plasmid with EcoRI revealed fragments of 230 and 1000 basepairs. The antibody used to screen the library was epitope selected against the fusion protein produced by the purified recombinant phage. The epitope-selected antibody, when incubated with Western blots of oviductal explant culture medium obtained from estradiol-treated baboons, recognized both the acidic and the basic variants of the glycoprotein. Northern blot hybridization with a 32P-labeled insert indicated that a single message of 2.8 kilobases was present in oviducts obtained from estradiol-treated baboons; no hybridization was detectable to RNA from oviducts obtained from progesterone-treated baboons. A mRNA of comparable size was also found in human, hamster, rabbit, and mouse oviduct tissue. In vitro translation of oviductal poly(A)+ RNA from an estradiol-treated baboon followed by immunoprecipitation with the polyclonal antibody against the acidic component indicated that the protein component of the oviductal glycoprotein had a mol wt of 66,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between gene expression and hybrid vigor in primary root tips of young maize (Zea mays L.) plantlets

To provide an insight into the molecular basis of heterosis, we investigated gene expression in primary root tips of a heterotic maize hybrid (B73 × Mo17) and its parental lines (B73 and Mo17). This analysis was carried out (i) by differential plaque hybridization of a recombinant cDNA library made to poly(A) RNA isolated from B73 × Mo17 primary root tips, and (ii) by comparing with two-dimensional gel electrophoresis proteins synthesized in vitro in the rabbit reticulocyte system by poly(A) RNA isolated, at different stages of development, from the three genotypes. The results showed that there are sets of proteins and mRNAs that are differentially synthesized and expressed in the F1 primary root tips in comparison to the parental lines. Moreover, results from the survey of 21 major in-vitrosynthesized polypeptide variants, from mRNAs of primary root tips of the parental lines and their F1 hybrid, indicated that in seven instances hybrid proteins translated in vitro were more abundant or possibly new. In most of the remaining cases, hybrid spots were similar in intensity to the same protein produced by one of the two parental lines.

Bullous Pemphigoid Antigen: cDNA Cloning, Cellular Expression, and Evidence for Polymorphism of the Human Gene

A human epidermal keratinocyte lambda gt11 recombinant cDNA library was screened with a 0.45-kb cDNA that was generated by polymerase chain reaction (PCR) amplification of a segment of human bullous pemphigoid (BP) antigen mRNA. The screen yielded five clones, the largest one, pcBPA-4, being 2.3 kb in size. The pcBPA-4 cDNA hybridized in Northern analyses with an approximately 9-kb mRNA from cultured keratinocytes, whereas no hybridization signal was detected with RNA from human skin fibroblast, fibrosarcoma HT-1080 cells, or amniotic epithelial WISH cell cultures. Nucleotide sequencing of pcBPA-4 revealed an open reading frame encoding a putative polypeptide of 447 amino acids. This polypeptide showed 88% homology with corresponding mouse BP antigen sequences, and a region of it was identical to a segment in previously published human BP antigen sequences. The 2.3-kb cDNA isolated here was different from a previously published human BP antigen cDNA, in that the open reading frame coded for 264 additional amino acids at the carboxyl end of the putative polypeptide. Known human BP antigen amino acid sequences, compared with mouse sequences, were predicted to be moderately divergent with a unit of evolutionary period (UEP) of 4.5 millions of years (MY). Southern hybridizations suggested that the BP antigen gene (BPAG1) is present as a single copy in the human genome. Southern analyses also revealed the presence of a StuI restriction fragment length polymorphism that can be used for linkage analyses to study the inheritance of a particular BPAG1 allele and a heritable cutaneous disorder affecting the basement membrane zone, such as epidermolysis bullosa.

Two human genes isolated by a novel method encode DNA-binding proteins containing a common region of homology

Two cDNAs encoding new DNA-binding proteins (Dbps) have been cloned using a human placenta λgt11 recombinant cDNA library and DNA fragments as probes. Hybrid proteins expressed by the λgt11 cDNA library were blotted onto nitrocellulose filters, and incubated with three different radio-labeled DNA probes containing the human epidermal growth factor (EGF) receptor enhancer or the human c- erbB-2 promoter. Two kinds of clones, named dbpA and dbpB, showed high affinities for the DNA probes. The comparison of the nucleotide and the deduced amino acid (aa) sequences between these two cDNAs indicated that 100 of 109 aa located in the central region of these two Dbps were identical. The dbpA and dbpB-coded proteins also had an affinity for other cDNA probes such as the human c- ski gene, but not for poly(dI-dC) · poly(dI-dC), suggesting that the sequence(s) recognized by the dbpA and dbpB-coded proteins may occur frequently, or that these proteins bind to DNA non-specifically in a different manner from that of histones. A simple method, described in this paper, can be used to isolate cDNA clones encoding Dbps. Strategies used for the detection of sequence-specific and non-specific Dbps are discussed.

Cloning of Full-length Elastin cDNAs from a Human Skin Fibroblast Recombinant cDNA Library: Further Elucidation of Alternative Splicing Utilizing Exon-specific Oligonucleotides

A human cDNA library was constructed utilizing RNA isolated from cultured skin fibroblasts. Recombinant clones containing elastin sequences were identified by plaque hybridizations with previously characterized human placental elastin cDNAs. Seven positive recombinant clones with inserts of approximately 3.2-2.2 kb were isolated. Characterization of the clones by restriction endonuclease analysis and dot-blot hybridizations with exon-specific synthetic oligonucleotides demonstrated considerable variability in the primary nucleotide sequence. Dideoxy nucleotide sequencing confirmed this finding. The variability is most likely a result of alternative splicing of exons from the primary elastin transcripts. The two largest clones contained approximately 1 kb of 3' untranslated sequence and approximately 2.2 kb of translated sequence encoding 730 amino acids. Six amino acids, encoded by exon 12A, have not been previously noted in human elastin cDNAs. In addition, these human skin fibroblast clones contained a 49 bp 5' untranslated sequence. These results demonstrate that there is considerable variability in the processed nucleotide sequence of the elastin mRNAs. These transcripts may code for isoforms of tropoelastin with different biologic properties.

Stage and strain specific expression of the tandemly repeated 90 kDa surface antigen gene family in Trypanosoma cruzi

A recombinant cDNA library constructed in the expression vector λgt11 using mRNA from the trypomastigote stage of Trypanosoma cruzi was screened with two monoclonal antibodies that have been shown to react with a 105 kDa and a 90 kDa surface antigen in trypomastigotes of the Peru and Y strains of T. cruzi. One recombinant λ phage, designated Tcc-20, was reactive to both monoclonals. The β-galactosidase/ T. cruzi hybrid protein encoded in Tcc-20 is recognized by the monoclonal antibodies and by serum antibodies from mice infected with strains of T. cruzi which contain the 90 kDa antigen. Antibodies immunoselected from serum of mice infected with the Peru strain by adsorption to Tcc-20 fusion protein react specifically with a 90 kDa polypeptide in trypomastigote but not epimastigote lysates of T. cruzi. The mRNA complementary to the DNA insert in Tcc-20 is present only in those stages and strains of T. cruzi which express the 90 kDa surface antigen. These characteristics are strong evidence that the T. cruzi DNA fragment cloned into Tcc-20 encodes a portion of the 90 kDa surface antigen. The gene(s) which encodes this polypeptide is shown to be present in approximately 20 copies per haploid genome and most, and possibly all, of the copies are found in a tandemly linked multigene family.

Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex.

To gain insight into factors associated with the excessive accumulation of beta-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the AD-specific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, we cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A)+ RNA from AD cortices was used for the preparation of lambda gt11 recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the beta-amyloid domain and corresponded to 75% of the coding region and approximately equal to 70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, we conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product.

Preparation of a recombinant cDNA library from poly(A+) RNA of the Alzheimer brain. Identification and characterization of a cDNA copy encoding a glial-specific protein

Studies were undertaken to assess the extent to which messenger RNA prepared from the postmortem Alzheimer's disease (AD) brain can be used for the successful preparation of a recombinant cDNA library. Initial experiments focused on the glial-specific marker glial fibrillary acidic protein (GFAP) since GFAP expression appeared to be a model for further studies on mRNAs that may continue to be expressed at high levels in the vicinity of lesioned sites in the AD brain. An AD cDNA library, prepared in the lambda gt11 expression vector system contained GFAP-specific recombinants. One of these was sequenced and the insert was shown to exhibit 88% homology with the similar sequence from mouse GFAP. As established by Northern blots, the size of the GFAP mRNA prepared from the routinely acquired postmortem AD cortex, approximately 2.7 kb, was the same as from a neurologically normal control brain. These results agree with earlier studies on GFAP mRNA from fresh mouse brain. The results demonstrate that in the postmortem AD brain, astroglial-specific mRNA remains sufficiently stable for molecular genetic analysis and may serve as a useful model for examining the genetic expression of mRNAs that may be related to the molecular pathogenesis and the etiology of AD.

Isolation of molecular hybridisation probes for early myeloid lineage RNAs.

Recombinant plasmid cDNA libraries representing the polyadenylated RNAs in the myeloid cell lines KG1 and ML1 have been constructed. The screening protocol has identified several clones which contain sequences homologous to RNAs expressed at high abundance in one or more of the myeloid cell lines KG1, ML1 and HL60. The relative abundances of RNAs homologous to three recombinants, pML15, pKG21 and pKGA/F5 were measured by an RNA dot hybridisation method in total RNAs isolated from peripheral blood leukocytes from leukaemic patients and normal individuals. High levels of these RNAs were observed mainly in ANLL and acute phase CML samples. The data suggest that these probes have the potential to sub-divide the ANLLs and to extend a molecular classification of the myeloid leukaemias.

Isolation of several cDNAs encoding yeast peroxisomal enzymes

Several candidate clones carrying partial cDNAs for yeast peroxisomal enzymes, such as catalase, carnitine acetyltransferase, isocitrate lyase, malate synthase and acyl-CoA oxidase, were efficiently isolated at a single plating from a phage λgt11 recombinant cDNA library prepared with poly(A)-rich RNA from an n-alkane-grown yeast, Candida tropicalis, with a mixture of antibodies against the respective purified enzymes. Among them, one candidate clone carrying partial cDNA for catalase was subcloned and subjected to nucleotide sequence analysis. We succeeded in determining that the amino acid sequence deduced from the nucleotide analysis included the sequences derived from the two peptide fragments obtained from the purified enzyme.

Genes Expressed in the Male Gametophyte of Flowering Plants and Their Isolation

Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period.