Isolation of two Drosophila melanogaster genes abundantly expressed in the ovary during vitelline membrane synthesis

Several genomic clones were isolated from a Drosophila library screened with cDNA prepared from abundant adult female mRNA. Cytoplasmic dot hybridizations have shown that the genes in all of these clones are expressed only in posteclosion (stages 8-14) follicles. One set of overlapping clones (lambda 20, lambda 28, and lambda 30) was localized by in situ hybridization to 66D, a previously described locus for chorion genes. Restriction mapping demonstrated that these clones contained chorion genes which had been isolated previously. Another clone, lambda 7, was mapped to chromosomal region 26A. This clone carries genes that hybridized to mRNA species similar or identical in size to the known chorion genes encompassed by lambda 28. Furthermore, one of these genes shows homology to the 66D chorion locus, apparently with the s18-1 gene. R-loop and S1-nuclease mapping indicated that lambda 7 contains two genes of 700-800 base pairs in length. Dot hybridization of cytoplasmic RNA from egg chambers demonstrated that these genes are expressed predominantly during stages 9 + 10, the time of vitelline membrane synthesis. Analysis of DNA extracted from embryos and various female tissues by dot hybridization showed that lambda 7 sequences are not amplified in the mature ovary. These results suggest that the two genes carried by lambda 7 and derived from region 26A may code for protein components of the vitelline membrane. In addition it appears that some evolutionary relatedness exists between one of these genes and a member of the chorion multigene family.     

The ornithine aminotransferase-encoding gene family of rat: cloning, characterization, and evolutionary relationships between a single expressed gene and three pseudogenes

As a first step towards understanding the molecular mechanisms through which the expression of the gene (OAT) encoding ornithine aminotransferase (OAT) is regulated in a tissue-specific manner, we have used a near full length OAT cDNA to isolate related sequences from a rat genomic DNA library. Twenty-one unique clones representing five contigs and spanning approximately 140 kb of genomic DNA were isolated and characterized. From these clones we have identified a single expressed OAT gene and three processed pseudogenes. The comparison of the EcoRI, BamHI, and HindIII fragments contained within these genomic clones with those detected in total genomic DNA by the cDNA probe suggests that essentially all of the OAT-related sequences in the rat genome have been isolated. Thus, the tissue-specific regulation of OAT gene expression appears to be effected through a single expressed gene. Data are presented which suggest that the OAT-1, OAT-2, and OAT-3 pseudogenes arose approximately 28.5, 7.3, and 25.1 Myr ago, respectively. Mutation rates are presented for each codon position of the expressed rat and human OAT genes. The region of the rat genome flanking the boundary of the OAT-3 pseudogene is of additional interest as it shares considerable identity to sequences contained within expressed genes and flanking other processed pseudogenes.     

Human chromosome 21-encoded cDNA clones

We have employed two strategies to isolate random cDNA clones encoded by chromosome 21. In the first approach, a cDNA library representing expressed genes of WA17, a mouse-human somatic cell hybrid carrying chromosome 21 as its sole human chromosome, was screened with total human DNA to identify human chromosome 21-specific cDNAs. The second approach utilized previously characterized single-copy genomic fragments from chromosome 21 as probes to retrieve homologous coding sequences from a human fetal brain cDNA library. Six cDNA clones on chromosome 21 were obtained in this manner. Two were localized to the proximal long arm of chromosome 21, two to the distal portion of the long arm, and one to the region of 21q22 implicated in the pathology of Down syndrome.     

Identification and isolation of transcribed human X chromosome DNA sequences

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short area of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.     

Identification of a Ferritin Light Chain Pseudogene Near the Glycerol Kinase Locus in Xp21 by cDNA Amplification for Identification of Genomic Expressed Sequences

We used cDNA amplification for identification of genomic expressed sequences (CAIGES) to identify genes in the glycerol kinase region of the human X chromosome. During these investigations we identified the sequence for a ferritin light chain (FTL) pseudogene in this portion of Xp21. A human liver cDNA library was amplified by vector primers, labeled, and hybridized to Southern blots ofEcoRIdigested human genomic DNA from cosmids isolated from yeast artificial chromosomes in the glycerol kinase region of Xp21. A 3.1-kb restriction fragment hybridized with the cDNA library, was subcloned and sequenced, and a 440-bp intronless sequence was found with strong similarity to the FTL coding sequence. Therefore, the FTL pseudogene that had been mapped previously to Xp22.3–21.2 was localized specifically to the glycerol kinase region. The CAIGES method permits rapid screening of genomic material and will identify genomic sequences with similarities to genes expressed in the cDNA library used to probe the cloned genomic DNA, including pseudogenes.     

Cloning and mapping of bovine ZFX gene to the long arm of the X-chromosome (Xq34) and homologous mapping of ZFY gene to the distal region of the short arm of the bovine (Yp13), ovine (Yp12–p13), and caprine (Yp12–p13) Y Chromosome

A part of mouse Zfy-2 sequence was synthesized and used to screen a genomic library of the spinous country-rat (Tokudaia osimensis spp., 2n = 45). An isolated clone had the C-terminal region of Zfy, which consisted of 1190 bp, encoded 336 amino acid residues, and harbored 11 out of 13 zinc finger motifs. With this as a probe, a bovine testis cDNA library was screened. Two ZFX clones were isolated and their sequences combined. The short sequence, lacking part of the 5′ upstream region, was amplified by PCR or RT-PCR, cloned, and sequenced. A full-length ZFX was constructed by combining these three sequences. The bovine ZFX consisted of 5328 bp and encoded 800 amino acid residues, which contained 13 zinc finger motifs. ZFX was used as a probe for fluorescence in situ hybridization and was mapped to Xq34, different from its previously reported site at Xq21-q231. A SINE (short interspersed nuclear element) sequence consisting of 188 bp was found close to the end of the 3′-untranslated region of ZFX. The SINE sequence hybridized to all bovine chromosomes. ZFY is highly homologous with ZFX and, as a result, ZFY could be mapped simultaneously. ZFY was mapped to the distal region of the short arm of the Y Chromosome (Chr) (Yp13), contradicting the previously reported position Yq1. Ovine and caprine ZFY were also mapped with bovine ZFX. Both were mapped to the distal region of the short arm of the Y Chr (Yp12-p13). Ovine ZFX was mapped to a region close to the centromere of the X Chr (Xq13). Received: 23 July 1997 / Accepted: 30 September 1997     

BAC contig development by fingerprint analysis in soybean.

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.     

Isolation, characterization, and regional mapping of microclones from a human chromosome 21 microdissection library.

Thirty-four unique-sequence microclones were isolated from a previously described microdissection library of human chromosome 21 and were regionally mapped using a cell hybrid mapping panel which consists of six cell hybrids and divides chromosome 21 into eight regions. The mapping results showed that the microclones were unevenly distributed along chromosome 21, with the majority of microclones located in the distal half portion of the long arm, between 21q21.3 and 21qter. The number of unique-sequence clones began to decrease significantly from 21q21.2 to centromere and extending to the short arm. This finding is consistent with those reported in other chromosome 21 libraries. Thus, it may be inferred that the proximal portion of the long arm of chromosome 21 contains higher proportions of repetitive sequences, rather than unique sequences or genes. The microclones were also characterized for insert size and were used to identify the corresponding genomic fragments generated by HindIII. In addition, we demonstrated that the microclones with short inserts can be efficiently used to identify YAC (yeast artificial chromosome) clones with large inserts, for increased genomic coverage for high-resolution physical mapping. We also used 200 unique-sequence microclones to screen a human liver cDNA library and identified two cDNA clones which were regionally assigned to the 21q21.3-q22.1 region. Thus, generation of unique-sequence microclones from chromosome 21 appears to be useful to isolate and regionally map many cDNA clones, among which will be candidate genes for important diseases on chromosome 21, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, and one form of epilepsy.     

Isolation of a zinc finger motif (ZNF75) mapping on chromosome Xq26.

We report here the partial characterization of a new human zinc finger (ZNF75) gene of the Kruppel type mapping to the long arm of the X chromosome. A cosmid clone was isolated from a library specific to the Xq24-qter region by hybridization to a degenerate oligonucleotide representing the link between two contigous fingers of the C2H2 type. The sequence of the pertinent cosmid fragments demonstrated five consecutive zinc finger motifs, all pertaining to the Kruppel family. A reading frame starting at least 75 amino acids before the first zinc finger and ending 11 amino acids after the last one was identified; comparison with other ZF genes suggests that this genomic fragment represents the carboxy-terminal exon of the gene. Homology of approximately 55% in the zinc finger region was detected with many zinc finger genes including mouse Zfp-35 and human ZFN7 cDNA clones. Mapping using a panel of sematic cell hybrids and chromosomal in situ hybridization localized the gene to Xq26, in a region not previously known to contain zinc finger genes.     

Structural analysis of the three vitellogenin genes in Drosophila melanogaster.

Genomic fragments coding for sequences expressed as abundant mRNA in female Drosophila melanogaster were isolated from a lambda library. Hybridization of these clones to polytene chromosomes. in situ, identified four which mapped to X chromosomal region 9A to 9B, the locus for yolk proteins 1 and 2 (Ypl,2) and two which mapped to 12A6-7 to 12D3, the locus for Yp3. These clones were mapped with restriction enzymes, and the coding regions and regions of homology determined by Southern blots probed with cDNA, 5'-end-labelled RNA and nick-translated DNA. Heteroduplex and R-loop mapping confirmed that three of the clones carried two genes separated by about 1.4 kb and oriented in opposite directions. Southern blots probed with cDNA made from alkali-hydrolyzed RNA showed that these genes had their 5' ends next to each other. All 3 genes show homology to each other and have a main coding region of about 1.3 kb, the approximate size for the mRNAs.     

Physical and cDNA mapping in the DBH region of human chromosome 9q34

Chromosome 9q34 has been extensively studied and mapped due to the presence of known disease genes, principally tuberous sclerosis 1 (TSC1), in this region. During the course of our mapping of this region we constructed a 555-kb contig beginning approximately 50 kb proximal to the dopamine-beta-hydroxylase (DBH) gene and extending, with one small deletion, distal to the D9S114 marker. The contig consists of 11 P1 clones, four PAC clones, one BAC clone and six cosmid clones and contains 27 new nonpolymorphic STSs. We have found the region to be unstable in P1, PAC and BAC cloning vehicles and have identified several deleted genomic clones. In addition, we have isolated and mapped the 3' portions of three putative genes located within or immediately distal to the DBH gene, including one large gene that runs on the opposite strand to DBH and utilizes portions of two DBH exons. The genomic clones of the contig, cDNAs and new STSs will be useful reagents for the further study and mapping of this region.     

A pseudogene homologous to mouse transplantation antigens: Transplantation antigens are encoded by eight exons that correlate with protein domains

We have isolated about 30 to 40 different BALB/c mouse sperm DNA genomic clones that hybridize to cDNA clones encoding proteins homologous to transplantation antigens. One of these clones (27.1) was selected for sequence analysis because it was polymorphic in Southern blot analyses of the DNAs from BALB/c and CBA mice. A fragment of 5.7 kilobases of this clone was completely sequenced and found to contain a pseudogene whose sequence is highly homologous to the sequences of known transplantation antigens. Pseudogene 27.1 is split into eight exons that correlate with the structurally defined protein domains of transplantation antigens. Using Southern blot hybridization on the DNAs of different inbred mouse strains, we mapped the pseudogene to the Qa-2,3 region, a part of the Tla complex on chromosome 17 that is adjacent to the major histocompatibility complex. The Qa-2,3 region encodes lymphoid differentiation antigens homologous to the transplantation antigens in size, in peptide map profiles and in their association with β2-microglobulin. These mapping studies suggest that gene 27.1 may be a pseudogene for either a Qa antigen or an as yet undefined transplantation antigen. Accordingly, we may have isolated genes encoding lymphoid differentiation antigens of the Tla complex as well as those encoding transplantation antigens among the 30 to 40 different genomic clones isolated from our sperm library.     

Two bovine genes for cytochrome c oxidase subunit IV: a processed pseudogene and an expressed gene

We have isolated and analyzed 17 clones from a bovine genomic library in phage lambda Charon28 probed with a bovine liver cDNA for cytochrome c oxidase subunit IV. Restriction enzyme mapping and Southern analysis indicated that these clones represent only two genomic regions. One region was shown by nucleotide sequencing to contain a subunit IV pseudogene of the processed type. The other class of clones contained the 5' region of a putative expressed gene; the region consists of two exons and two introns, with one exon encoding exclusively the domain representing the presequence present on newly synthesized subunit-IV polypeptides. Genomic Southern analysis indicated that these two clones probably represent the only sequences in the bovine nucleus that share nucleotide sequence identity with the liver subunit IV cDNA when utilizing moderately stringent hybridization conditions.