Analysis of the size of the carcinoembryonic antigen (CEA) gene family: isolation and sequencing of N-terminal domain exons

Five members of the human CEA gene family [human pregnancy-specific beta 1-glycoprotein (PS beta G); hsCGM1, 2, 3 and 4] have been isolated and identified through sequencing the exons containing their N-terminal domains. Sequence comparisons with published data for CEA and related molecules reveal the existence of highly-conserved gene subgroups within the CEA family. Together with published data eleven CEA family members have so far been determined. Apart from the highly conserved coding sequences, these genes also show strong sequence conservation in their introns, indicating a duplication of whole gene units during the evolution of the CEA gene family.     

Exon-intron organization of a gene for pregnancy-specific beta 1-glycoprotein, a subfamily member of CEA family: implications for its characteristic repetitive domains and C-terminal sequences

A fragment of human gene for pregnancy-specific beta 1-glycoprotein(s), recently identified CEA family member(s), has been cloned. Analyses of nucleotide and deduced amino acid sequences revealed that it carried, from 5' to 3' direction, exons IA, IB, IIA, IIB, C3, C1 and C2, the first four encoding peptides distinct from but highly similar to domains of PS beta Gs. The lack of consensus 3' splice site sequence ahead of IB indicated that it was an abortive exon, which would explain the peculiar domain construction of PS beta Gs, i.e. N-IA-IIA-IIB-C1, 2 or 3. Apparently, the multiple C-terminal sequences for a PS beta G were generated by alternative splicing among C1, C2 and C3 exons. Furthermore, sequences which overlapped partly with Cexons, were found to be similar to parts of 3'-UTR of CEA and NCA, indicating further the close relationship of CEA/NCA and PS beta G subfamily genes.     

Expression of CEA-related genes in the first trimester human placenta

Eight cDNA clones, closely related to the carcino-embryonic antigen gene family, have been isolated from a cDNA library representing genes expressed in the first trimester human placenta. Sequence analysis of one clone shows it to be a pregnancy-specific beta 1-glycoprotein (PS beta G) closely related to three other PS beta G cDNA recently characterised from a term placenta library. The protein encoded by the cDNA is predicted to be less high glycosylated than those reported previously and differs markedly in the C-terminal sequence. The 3' untranslated region of the cDNA is very similar to the equivalent region of beta 1-glycoprotein PS beta G E except that it contains the 12bp repeat sequence found flanking the Alu sequence in CEa and an additional 67bp of sequence that appears to be derived from CEA.     

The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of pregnancy-specific beta 1-glycoprotein (PS beta G)

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific beta 1-glycoprotein (PS beta G). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8-83.5% to those of the PS beta G members so far reported, indicating PS34 is a new member of PS beta G and also of the carcinoembryonic antigen gene family.     

Identification and mapping on human chromosome 19 of chromosome-specific repeated element preferentially binding with nuclear matrix]

From a library of sequences binding preferentially to nuclear matrix (matrix attachment regions, MARs), a fragment of about 300 bp in length (CEA (carcinoembryonic antigen)-MAR) was isolated and characterized. The CEA-MAR sequence was found in more than ten loci of chromosome 19 containing elements similar to genes of the CEA family. No sequences of this group were found on other human chromosomes. Two CEA-MAR-containing loci were sequenced, and sequences for another seven loci were found in GenBank. A comparative analysis of CEA-MARs and the flanking sequences is reported. Based on the sequence of the CEA-containing chromosome 19 loci, a hypothetical model of the domain structure of a 2-Mb chromosome region was constructed and the mutual arrangement of CEA-MARs and genes of CEA family was elucidated. The CEA-MARs were located 5-20 kb downstream of the CEA genes. These results suggest that the duplication unit of the CEA family may coincide with chromatin domains containing these genes.     

A pregnancy-specific beta 1-glycoprotein, a CEA gene family member, expressed in a human promyelocytic leukemia cell line, HL-60: structures of protein, mRNA and gene

Both genomic and cDNA clones encoding a precursor for a pregnancy-specific beta 1-glycoprotein (PS beta G) belonging to the CEA family, expressed in a human promyelocytic leukemia cell line, HL-60, have been isolated and the entire primary structure of the precursor is deduced. The 335-AA precursor has a 34-AA signal peptide followed by domains of N, IIA, IIB and C, which are encoded by separate exons. The genomic sequence contains extra exons IA and IB between exons N and IIA. Apparently, exon IA is excluded from the mRNA by alternative splicing while IB is a pseudo-exon having a stop codon formed by a deletion of dinucleotide in the middle of the sequence. This provides another mechanism to render exon IB abortive and is different from that we reported for another PS beta G (Biochem. Biophys. Res. Comm. (1988) 156, 68-77).     

Characterization of cDNAs of the human pregnancy-specific beta 1-glycoprotein family, a new subfamily of the immunoglobulin gene superfamily

Three highly homologous cDNAs encoding human pregnancy-specific beta 1-glycoprotein (SP1) were isolated from a human placental cDNA library. These cDNAs share greater than 90% nucleotide homology in their coding sequences, and greater than 79% of the encoded amino acids are homologous. Proteins encoded by these cDNAs are very similar to members of the carcinoembryonic antigen family and contain repeating domains, conserved disulfide bridges, and beta-sheet structure typical of the immunoglobulin gene superfamily. However, the high degree of sequence homology and relatively lesser degree of glycosylation among the SP1 proteins suggest that they exist as a unique family instead of being members of the CEA family. Both soluble and potentially membrane-bound forms of SP1 proteins were present in the placenta. Northern blot analysis using specific probes confirmed the expression of multiple mRNA species in human term placenta.     

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.     

Effects of sodium butyrate on the synthesis of human pregnancy-specific beta 1-glycoprotein

Human pregnancy-specific beta 1-glycoprotein (PS beta G) is a polymorphic placental protein which shows strong sequence similarity with the oncofetal protein, carcinoembryonic antigen. To better understand the role of PS beta G in pregnancy, we examined its synthesis and regulation in placental fibroblasts, which had been shown to express the PS beta G gene. The major placental PS beta G is a 72-kDa glycoprotein, while the major fibroblast PS beta G is a 62-kDa species. Administration of sodium butyrate to these fibroblasts slightly stimulated the synthesis of the 62-kDa species but markedly increased the production of two additional PS beta Gs of 72 and 48 kDa. The similarity between the PS beta Gs synthesized by butyrate-treated fibroblasts and human placenta was confirmed by cell-free protein synthesis. Poly(A)+ RNA from butyrate-treated fibroblasts and placenta directed the synthesis of two polypeptides of 48 and 36 kDa, which form the polypeptide backbone of the 72- and 48-kDa glycoproteins. Moreover, the predicted molecular weights of PS beta Gs encoded by the two types of PS beta G cDNA clones were 48,000 and 36,000. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity at the 5' region (designated PSG-5') but differ in sequences at their 3' regions. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases from placental fibroblasts. Butyrate increased the steady-state levels of all three mRNAs. Ribonuclease protection analysis showed that butyrate increased the PS beta G mRNAs containing the PSG-5' or PSG93-specific sequence to approximately 20% of human placental levels. However, unlike human term placenta, which predominantly expressed PS beta G mRNAs with 3'-sequences similar to PSG16/PSG93, the butyrate-treated fibroblasts expressed roughly equal levels of PS beta G mRNAs with the PSG16/PSG93-3' and PSG95-3' ends. All PS beta G cDNAs identified encode proteins with distinct carboxyl termini, suggesting that the composition of the 72-kDa species in placenta and butyrate-treated fibroblasts is likely to be different. Placental fibroblasts provide a unique model for the study of the mechanisms responsible for the differential expression of the PS beta G gene.     

Characterization of pregnancy-specific beta 1-glycoprotein synthesized by human placental fibroblasts

We have previously demonstrated that human placental fibroblasts produce a pregnancy-specific beta 1-glycoprotein (PS beta G) immunologically indistinguishable from placental PS beta G. This was confirmed by the immunocytochemical localization of PS beta G in these fibroblasts. In addition, placental fibroblasts contain all three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases which hybridize with the three PS beta G cDNAs (PSG16, PSG93, and PSG95) identified, although at 1.4-2.5% of the levels in human term placenta. The major PS beta G species synthesized by placental fibroblasts is a 62K glycopolypeptide formed from a 58K intracellular precursor polypeptide. However, the PS beta G species found in human placenta are one major glycoprotein of 72K and two minor ones of 64K and 54K. Poly(A)+ RNA from placental fibroblasts directed the synthesis of two polypeptides of 48K and 46K (major), whereas, poly(A)+ RNA from human placenta directed the synthesis of higher levels of four polypeptides of 50 K, 48 K (major), 46 K, and 36 K. Thus, the major PS beta G species found in fibroblasts and human placenta differ. The carbohydrate side-chains are essential for the stability of fibroblast PS beta G, because PS beta G synthesis in these fibroblasts could not be detected in the presence of tunicamycin, a protein glycosylation inhibitor which did not affect PS beta G mRNA expression. Our finding that a variant PS beta G species is produced in placental fibroblasts raises the possibility that the authentic placental PS beta G species may have different functions.     

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.     

The human protein S locus: identification of the PS alpha gene as a site of liver protein S messenger RNA synthesis

The protein S locus, situated on chromosome 3, consists of two protein S genes. Here, we report the cloning and complete nucleotide sequence of the 3'-untranslated region of the two genes designated PS alpha and PS beta. Both regions span approximately 1,200 nucleotides. They show a high degree (-97%) of homology, with deviations caused by small deletions, insertions and point mutations. Comparison of PS alpha and PS beta with the reported protein S liver cDNAs, shows that the latter all originate from the PS alpha gene. The PS alpha gene therefore is marked as the major site of synthesis of liver protein S mRNA. Sequence comparison with the bovine protein S cDNA reveals that the PS beta gene has accumulated a few more mutations than the PS alpha gene since duplication of the ancestral protein S gene that seems to have occurred recently during primate evolution.     

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.     

Identification of three new genes and estimation of the size of the carcinoembryonic antigen family.

Using carcinoembryonic antigen (CEA) subgroup-specific degenerate PCR primers, we have identified three new CEA gene family member L/N exons (CGM9, CGM10, and CGM11) and all previously reported L/N exons of the CEA subgroup (CEA, BGP, NCA, CGM1, CGM2, CGM6, CGM7, and CGM8). This suggests that the CEA subgroup contains 11 genes. CGM9, CGM10, and CGM11 seem to be pseudogenes. A deletion of an asparagine in CGM9 results in loss of a glycosylation site, which is conserved throughout the CEA gene family. We have previously suggested the number of genes in the pregnancy-specific glycoprotein (PSG) subgroup to be 11, which together with this study indicates that the CEA gene family contains 22 genes in all. Parsimony analysis of the CEA subgroup interrelationships suggests that CGM7 occupies the most primitive position within the CEA subgroup, being a sister group to the rest. CEA, BGP, NCA, and CGM1 form a fairly well-supported group within the CEA subgroup.     

The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of Pregnancy-Specific β1-Glycoprotein (PSβG)

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific β₁-glycoprotein (PSβG). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8–83.5% to those of the PSβG members so far reported, indicating PS34 is a new member of PSβG and also of the carcinoembryonic antigen gene family.