Long-range chromosomal mapping of the carcinoembryonic antigen (CEA) gene family cluster

A long-range physical map of the carcinoembryonic antigen (CEA) gene family cluster, which is located on the long arm of chromosome 19, has been constructed. This was achieved by hybridization analysis of large DNA fragments separated by pulse-field gel electrophoresis and of DNA from human/rodent somatic cell hybrids, as well as the assembly of ordered sets of cosmids for this gene region into contigs. The different approaches yielded very similar results and indicate that the entire gene family is contained within a region located at position 19q13.1-q13.2 between the CYP2A and the D19S15/D19S8 markers. The physical linkage of nine genes belonging to the CEA subgroup and their location with respect to the pregnancy-specific glycoprotein (PSG) subgroup genes have been determined, and the latter are located closer to the telomere. From large groups of ordered cosmid clones, the identity of all known CEA subgroup genes has been confirmed either by hybridization using gene-specific probes or by DNA sequencing. These studies have identified a new member of the CEA subgroup (CGM8), which probably represents a pseudogene due to the existence of two stop codons, one in the leader and one in the N-terminal domain exons. The gene order and orientation, which were determined by hybridization with probes from the 5' and 3' regions of the genes, are as follows: cen/3'-CGM7-5'/3'-CGM2-5'/5'-CEA-3'/5'-NCA-3'/5'-CGM1- 3'/3'-BGP-5'/3'- CGM9-5'/3'-CGM6-5'/5'-CGM8-3'/PSGcluster/qter.     

Order and genomic distances among members of the carcinoembryonic antigen (CEA) gene family determined by fluorescence in situ hybridization.

Fluorescence in situ hybridization was used to establish the order of, and to estimate genomic distances among, members of the carcinoembryonic antigen (CEA) and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19. Fluorescence in situ hybridization to metaphase chromosomes localized the PSG subgroup telomeric to the CEA subgroup. Cosmid clones containing sequences for individual genes in the CEA and PSG subgroups were also hybridized to human sperm pronuclear and somatic interphase nuclear chromatin targets. The mapping results lead to the gene order cen-CGM7-CEA-NCA-CGM1-BGP-CGM9-CGM8-PSG-te l. The genomic distances between selected pairs of gene family members were estimated from the physical distances between hybridization sites measured in pronuclei. The CEA-PSG gene family region is estimated to span 1.1 to 1.2 Mb.     

Assembly and analysis of cosmid contigs in the CEA-gene family region of human chromosome 19.

The carcinoembryonic antigen (CEA)-like genes are members of a large gene family which is part of the immunoglobulin superfamily. The CEA family is divided into two major subgroups, the CEA-subgroup and the pregnancy-specific glycoprotein (PSG)-subgroup. In the course of an effort to develop a set of overlapping cosmids spanning human chromosome 19, we identified 245 cosmids in a human chromosome 19 cosmid library (6-7X redundant) by hybridization with an IgC-like domain fragment of the CEA gene. A fluorescence-based restriction enzyme digest fingerprinting strategy was used to assemble 212 probe-positive cosmids, along with 115 additional cosmids from a collection of approximately 8,000 randomly selected cosmids, into five contigs. Two of the contigs contain CEA-subgroup genes while the remaining three contigs contain PSG-subgroup genes. These five contigs range in size from 100 kb to over 300 kb and span an estimated 1 Mb. The CEA-like gene family was determined by fluorescence in situ hybridization to map in the q13.1-q13.2 region of human chromosome 19. Analysis of the two CEA-subgroup contigs provided verification of the contig assembly strategy and insight into the organization of 9 CEA-subgroup genes.     

The pregnancy-specific glycoprotein family of the immunoglobulin superfamily: Identification of new members and estimation of family size

The members of the carcinoembryonic antigen (CEA)/pregnancy-specific glycoprotein (PSG) gene family have a characteristic N-terminal domain that is homologous to the immunoglobulin variable region. We have estimated the size of the PSG subfamily by identification of N-domain exons from isolated genomic clones and from total genomic DNA through PCR amplification and DNA sequence determination. The PSG subfamily contains at least 11 different genes. For 7 of these, two DNA sequences differing from each other in 1 to 4 nucleotides were detected. Most likely, they represent different alleles. They are PSG1, PSG2, PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG11, PSG12, and PSG13. Six of the N-domain sequences described here are new. All of the PSGs except PSG1, PSG4, and PSG8 contained the arginine-glycine-aspartic acid sequence at position 93-95 corresponding to the complementarity determining region 3 of immunoglobulin. Parsimony analysis of 24 CEA and PSG sequences using 12 members of the immunoglobulin gene superfamily as outgroups to root the family tree shows that the N-domain of the CEA group genes evolved in one major branch and the PSG group genes in the other.     

Human pregnancy-specific beta 1-glycoproteins are coded within chromosome 19.

We have isolated and characterized cDNAs that code for apoproteins having amino acid sequences highly similar to pregnancy-specific beta 1-glycoproteins (PS beta G). cDNAs coding for PS beta Gs, as well as the cDNA clone reported here, are members of the carcinoembryonic antigen (CEA) gene family. The previous localization of CEA-related genes to human chromosome 19, and the high level of DNA sequence conservation in the CEA family, suggested that the PS beta G genes are also located on this chromosome. We demonstrate here that chromosome 19 is indeed the site of PS beta G sequences. Our finding is in contrast to the recently reported indication that pregnancy-specific glycoproteins are encoded in chromosomes X and 6.     

A restriction map of the ribosomal RNA genes and the short single-copy DNA sequence of the pearl millet chloroplast genome

The chloroplast rDNA genes of pearl millet (Pennisetum americanum) have been cloned and physically mapped. The chloroplast genome of the pearl millet contains two identical rRNA genes located on DNA sequences that are inverted with respect to one another and separated by 12 kb of single-copy DNA. The rRNA genes were positioned on a restriction endonuclease map by using as hybridization probes specific cloned rDNA sequences from the chloroplast DNA of the alga Euglena gracilis. The 16S and 23S rRNA genes were shown to be approx. 2 kb from one another, and the 5S RNA gene is immediately adjacent to the 23S tRNA gene.     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.     

Structural similarity of the HLA-DQ region in DQ3 and DQ4 haplotypes and structural diversity of the HLA-DQ region in HLA-DR7 haplotypes.

Genomic DNA obtained from a B lymphoblastoid cell line was digested with appropriate restriction endonuclease and hybridized with several probes specific for genes encoding HLA-DQ. Southern hybridization with a DQA1 3'untranslated (UT) region probe showed DQ2-type hybridization pattern in DR7DQ3 haplotype. On the contrary, DQB1 3'UT probe showed DQ3-type pattern in the same haplotype. Gene cloning and DNA sequencing analysis revealed a repetitive sequence, (TG)19, between DQA1 and DQB1 gene in the DR7DQ3 haplotype. These results suggest that a recombination event has occurred near this potential Z-DNA structure in the haplotype, DR7DQ3. The 3'UT region probes of DQA1 and DQB1 genes failed to detect restriction fragment length polymorphism (RFLP) differences between DR4DQ3 and DR4DQ4 haplotypes in this experiment, suggesting that the gene structure between DQA1 and DQB1 is conserved in these haplotypes.     

The number of large ribosomal RNA genes in Leptospira interrogans and Leptospira biflexa

We determined the number of large ribosomal RNA genes in five strains of Leptospira by hybridization of 15 restriction endonuclease digests of genomic DNA to the [32P]-labeled fragment of 23s rRNA gene. Almost all the restriction gels gave two radioactive bands. The conclusion from these results is that there are at least two rRNA genes in these leptospiral strains. Furthermore, the hybridization patterns of L. icterohaemorrhagiae strains Ictero No. I and RGA are almost identical. The number of rRNA genes and taxonomic relationships of these leptospires were discussed.     

Localization of human alpha 1 acid glycoprotein genes to 9q31----34.1

There is evidence for more than one alpha 1 acid glycoprotein (alpha 1 AGP) gene, and they all appear to be in close proximity. In situ hybridization of the cloned human cDNA p alpha 1AGP-2 to human chromosomes indicates that the alpha 1AGP genes are located between bands q31 and q34.1 on chromosome 9. This finding is in agreement with the previous assignment of the locus for alpha 1AGP to a linkage group with ABO and AK on chromosome 9.     

Isolation of yeast tRNALeu genes. DNA sequence of a cloned tRNALeu3 gene.

A library of cloned yeast DNA fragments generated by digestion of yeast DNA with the restriction endonuclease Bam HI has been screened by colony hybridization to total yeast [32P]tRNA. Four hundred colonies carrying yeast tRNA genes were isolated. By hybridization to 125I-tRNALeu3, we have isolated from this collection 14 colonies carrying fragments containing yeast tRNALeu genes. The size of the yeast Bam HI inserts ranged from 2.45 x 10(6) to 14 x 10(6) daltons. One of these fragments was mapped in detail by restriction endonuclease digestion and hybridization to 125I-tRNALeu3. The presence of a tRNALeu3 gene was confirmed by DNA sequence. The results indicate that the tRNALeu3 coding region is not co-linear with the tRNALeu3. An intervening tract of 33 base pairs interrupts the coding sequences 1 base pair past the anticodon coding region. The putative structure of a tRNALeu3 precursor is deduced in which the anticodon base pairs with residues from the intervening sequence.     

Hemoglobin switching in sheep. Cloning and characterization of the beta A and beta-like embryonic globin genes from genomic DNA

Genomic DNA from a fetal sheep homozygous for the beta A gene was used to construct a library of one million cloned DNA fragments using the bacteriophage vector, Charon 4A. Screening of 150,000 plaques from this library using radioactive beta-globin gene sequences resulted in the isolation of two recombinant bacteriophage containing globin genes. One of these, S beta AG-21, contains the complete adult beta A-globin gene as demonstrated by hybridization and restriction endonuclease analysis. In common with adult globin genes from other species, the beta A gene contains small (105 base pairs) and large (900 base pairs) intervening sequences. The second recombinant bacteriophage, SG-4, contains a complete embryonic beta-like globin gene which is expressed in the sheep embryo as demonstrated by hybridization analysis with cDNA made from sheep embryonic globin mRNA. Although differing in its restriction endonuclease map from the adult beta-globin genes, SG-4 appears to contain a large intervening sequence of at least 750 base pairs in length. Finally, preliminary evidence is discussed which indicates that a Pvu II site just 5' to the Cap site may be a common feature of sheep globin genes.